Methods and compositions related to T-cell activity

ABSTRACT

Embodiments concern methods and composition related to anergic T-cells in patients, such as cancer patients. T cell anergy is a hyporesponsive state induced by TCR engagement in the absence of costimulation (Schwartz, 2003). Anergy induction was initially observed in vitro using chemically-fixed antigen presenting cells (APCs). Subsequently, it was found that anergy could be induced by immobilized anti-CD3 mAb or calcium ionophores (such as ionomycin) in vitro, and by superantigen and soluble antigenic peptide in vivo. Indirect evidence has suggested that T cell dysfunction in the tumor microenvironment and establishment of transplant tolerance is partially due to T cell anergy (Gajewski et al., 2011).

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a national phase application under 35 U.S.C. § 371of International Application No. PCT/US2014/029173, filed Mar. 14, 2014,which claims the benefit of priority to U.S. Provisional PatentApplication Ser. No. 61/794,535, filed Mar. 15, 2013. The entirecontents of each of the above-referenced disclosures are specificallyincorporated herein by reference without disclaimer.

GOVERNMENTAL RIGHTS

The invention was made with government support under Grant Nos. R01CA161005, R01 AI080745, and R21 AI79373 awarded by the NationalInstitutes of Health. The government has certain rights in the invention

BACKGROUND OF THE INVENTION 1. Field of the Invention

The present invention relates generally to the field of medicine. Moreparticularly, it concerns the EGR2 transcriptome and targets that areused to diagnose or modified to treat T cell anergy and cancer.

2. Background

T cell anergy is a hyporesponsive state induced by TCR engagement in theabsence of costimulation (Schwartz, 2003). Anergy induction wasinitially observed in vitro using chemically-fixed antigen presentingcells (APCs). Subsequently, it was found that anergy could be induced byimmobilized anti-CD3 mAb or calcium ionophores (such as ionomycin) invitro, and by superantigen and soluble antigenic peptide in vivo.Indirect evidence has suggested that T cell dysfunction in the tumormicroenvironment and establishment of transplant tolerance is partiallydue to T cell anergy (Gajewski et al., 2011). T cell anergy is mainlycharacterized by the non-responsive state and multiple TCR signalingdefects, of which, blunted Ras/MAPK activation has been consistentlyobserved both in vitro and in vivo anergy models (Zheng et al., 2008).Further studies elucidated that the TCR signaling defects are due topresence of so called “anergy-associated factors”, which arespecifically synthesized upon anergy induction. Severalanergy-associated factors have been identified, including diacylglycerolkinase-α and -ζ (DGK-α and DGK-ζ); the E3 ubiquitin ligases Cbl-b,GRAIL, and Itch; Deltex 1 (Dtx1); and the anti-proliferative proteinTob1. In particular, the inventors and others have demonstrated thatDGK-α and DGK-ζ attenuate Ras/MAPK signaling by depleting diacylglycerol(DAG) (Olenchock et al., 2006; Zha et al., 2006).

The mechanisms leading to the generation of the anergy-associatedfactors have been gradually understood. TCR engagement alone activatesthe calcium/calcineurin/NFAT pathway out of proportion to AP1activation, resulting in the upregulation of early growth response gene2 and 3 (Egr2 and Egr3). Egr2 and Egr3 are transcriptional factorscontaining zinc finger domains (Chavrier et al., 1988; Patwardhan etal., 1991). The inventors and others have conducted gene-array analysescomparing anergic versus non-anergic T cells, and found that Egr2 ishighly upregulated 2-3 hours after anti-CD3 treatment, which is reducedby calcineurin inhibitor cyclosporine A (Harris et al., 2004; Safford etal., 2005; Zha et al., 2006). The expression of Egr2 in anergic cellswas of interest because the promoter region of the DGK-α gene containedan Egr2 binding site (Zheng et al., 2012). Forced-expression of Egr2 hasbeen reported to suppress T cell activation as demonstrated bydiminished IL-2 production and proliferation (Harris et al., 2004;Safford et al., 2005). Conversely, Egr2-deleted T cells are largelyresistant to anti-CD3-induced anergy in vitro with restored IL-2production and Erk phosphorylation (Zheng et al., 2012). Similarfindings were observed in superantigen staphylococcal enterotoxin B(SEB)-induced anergy in vivo as well. Furthermore, conditionalEgr2-deficient mice demonstrated enhanced anti-tumor immunity. Thenecessity of Egr2 in T cell anergy is partially due to its involvementin the regulation of most identified anergy-associated genes. ChIPassays and qRT-PCR confirmed that Egr2 interacts with and directlypromotes the transcription of DGK-α, DGK-ζ, Cbl-b, Itch, Dtx1, and Tob1in anergic cells.

Despite these advances in T cell anergy, knowledge about the anergicphenotype remains incomplete for several reasons. First, surface markersthat might be used to identify anergic T cells are lacking Second, ithas been unclear teleologically why T cells being subjected toanergy-inducing conditions are not simply deleted from the repertoire,in order to eliminate T cells of undesired specificities. Therefore,there remains a need to understand how T cell anergy is implemented,particularly as it relates to understanding how some tumors may be lesssusceptible than others to respond to immunotherapy.

SUMMARY OF THE INVENTION

Methods and compositions are provided for identifying T cell anergy andtreating a patient depending on whether T cells are anergic.

In certain aspects, methods are provided that involve a subject or apatient including, for example, the following: methods for evaluatingT-cell anergy in a patient, methods for treating a patient with animmunotherapy, methods for evaluating an immune response, or methods forpromoting a T-cell response in a subject. In any such methods, thesubject or patient may be any subject or patient in need of suchevaluation, treatment, or modulation of a cellular response. Forexample, the subject or patient may be a cancer subject or cancerpatient. In other aspects, the subject or patient is a subject orpatient having a chronic viral infection (e.g., HIV, hepatits C, and thelike). Moreover, a subject or patient may be a mammal, including ahuman.

In certain aspects, the disclosed methods and compositions are directedto nucleic acid molecules, such as those that reduce the expression oractivity of a gene including, for example, the T-cell anergy geneslisted in Table 2. However, it will be appreciated by one of ordinaryskill in the art that such methods or compositions may alternativelytarget the product of the particular gene of interest. Thus, in any ofthe disclosed methods and compositions, it is contemplated that theproduct of a gene of interest may be modulated by using a molecule thatspecifically targets or binds to a polypeptide or protein product of thegene of interest (such as one or more of the genes listed in Table 2).Examples of such molecules that target gene products include, but arenot limited to, polypeptides, antibodies, antibody fragments, aptamers,or small molecules.

Certain embodiments are directed to T-cell anergic genes, such as thoselisted in Table 2. Any such embodiments directed to T-cell anergic genesmay involve one or more of the genes included Table 2, or mayalternatively involve one or more of the genes included in the tableprovided in FIG. 14.

In certain embodiments, there are method for evaluating T-cell anergy ina patient comprising: a) measuring in T-cells from the patient anincrease in expression level(s) of one or more T-cell anergic genes inTable 2 compared to a reference or control level of expression innon-anergic T-cells; and, b) identifying the patient as having anergicT-cells.

In further embodiments, there are methods for evaluating T-cell anergyin a patient is provided, the method comprising: measuring in T-cellsfrom the patient expression level(s) of one or more T-cell anergic genesin Table 1; b) comparing the level to a reference or control level ofexpression T-cells identified as anergic; and c) identifying the patientas having anergic T-cells if the T-cells are determined to have anexpression level that is decreased or less than about two-fold increasedexpression compared to a reference or control level of expression in theT-cells identified as anergic.

Also provided are methods for evaluating T-cell anergy in a patientcomprising: a) measuring in T-cells from the patient an increase inexpression level(s) of one or more T-cell anergic genes in Table 2compared to a reference or control level of expression in non-anergicT-cells; and, b) identifying the patient as having anergicT-cells—wherein measuring an increase in expression comprises measuringan increase in protein expression.

In some embodiments, at least or at most 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47,48, 49, or 50 genes in Table 2 (or any range derivable therein) have atleast about or at most about a two-, three-, four-, five-, 6-, 7-, 8-,9-, 10-, 12-, 15-, 20-, 25-, 30-, 35-, 40-, 45-, 50-, 60-, 70-, 80-,90-, 100-fold increase in expression (or any range derivable therein) ascompared to a reference or control level. In some aspects, the increasein expression is at about a five-fold increase. In further aspects, theT-cell anergic genes that are measured include a cytokine; or the T-cellanergic genes that are measured include a cell surface receptor (whichmay include, in some embodiments, wherein the cell surface receptor isSemaphorin 7A, Class-I-MHC-restricted T cell associated molecule(Crtam), lymphocyte-activation gene 3 (Lag3), 4-1BB and/or Nrn1); or theT-cell anergic genes that are measured include an intracellular protein(which may include, in some embodiments, wherein the intracellularprotein is Bim, Nrgn, and/or Crabp2). In further aspects, any of theabove methods for evaluating T-cell anergy in a patient may furthercomprise isolating T-cells from the patient (which may include, in someembodiments, wherein T-cells are isolated from a blood sample or a tumoror cancer sample from the patient—with the understanding that a cancersample includes cancer cells or cells suspected of being cancerous). Infurther aspects, any of the above methods for evaluating T-cell anergyin a patient may further comprise obtaining a blood sample or a tumor orcancer sample from the patient. In further aspects, any of the abovemethods for evaluating T-cell anergy in a patient may be wherein:measuring an increase in expression comprises measuring an increase inRNA expression (which may be, for some embodiments, wherein an increasein RNA expression is measured comprising employing a hybridizationand/or amplification assay). In additional aspects, any of the abovemethods for evaluating T-cell anergy in a patient may be whereinmeasuring an increase in expression comprises generating a cDNA of themRNA transcript encoded by T-cell anergic gene. In related aspects, anyof the above methods for evaluating T-cell anergy in a patient mayfurther comprise amplifying cDNA corresponding to a T-cell anergic gene(which may be, for some embodiments, wherein amplifying cDNA comprisesincubating the cDNA with one or more primer sets specific to the cDNAand performing polymerase chain reaction (PCR)).

In some aspects, the method for evaluating T-cell anergy in a patientmay be wherein one or more binding proteins specific for a polypeptideencoded by a T-cell anergic gene is used (which may be, for someembodiments, wherein the one or more specific binding proteinscomprise(s) all or part of an antibody specific for a polypeptideencoded by a T-cell anergic gene, where the method may or may notfurther comprise employing a quantitative immunoassay to measure proteinexpression). In related aspects, any of the above methods for evaluatingT-cell anergy in a patient may be wherein expression is measured usingan array or microarray.

In some embodiments of above methods, method for evaluating T-cellanergy in a patient is provided, the method comprising: measuring inT-cells from the patient expression level(s) of one or more T-cellanergic genes in Table 1; b) comparing the level to a reference orcontrol level of expression T-cells identified as anergic; and c)identifying the patient as having anergic T-cells if the T-cells aredetermined to have an expression level that is decreased or less thanabout two-fold increased expression compared to a reference or controllevel of expression in the T-cells identified as anergic—is whereinmeasuring an increase in expression comprises measuring an increase inprotein expression (which may be, for some embodiments, wherein: one ormore binding proteins specific for a polypeptide encoded by a T-cellanergic gene is used; and/or the one or more specific binding proteinscomprises all or part of an antibody specific for a polypeptide encodedby a T-cell anergic gene, and/or further comprises employing aquantitative immunoassay to measure protein expression). In someaspects, an above method may be wherein expression is measured using anarray or microarray. It is contemplated that a measured level and areference or control level of the T-cell anergy gene may be normalized.In some embodiments, the expression level of a particular gene may beused for both the measured level and the control or reference level,though they need not be.

In some embodiments, a method for treating a patient with immunotherapyis provided, the method comprising administering immunotherapy to thepatient after the patient is identified as having non-anergic T-cells.In some aspects, this method may be wherein: the patient is identifiedas having non-anergic T-cells by measuring increased expression level(s)of one or more T-cell anergic genes in Table 2 compared to theexpression level to a reference or control level of expression innon-anergic T-cells; or the patient is identified as having non-anergicT-cells by measuring expression level(s) of one or more T-cell anergicgenes in Table 2 and comparing the expression level to a reference orcontrol level of expression in anergic T-cells. In related aspects, anabove method may be wherein: the immunotherapy comprises a cell-basedimmunotherapy; the immunotherapy may comprise antibody therapy, avaccine (such as a cancer vaccine), a checkpoint inhibitor, cytokineIL-2, or an adoptive T cell therapy.

In further embodiments, a method for treating a patient is provided, themethod comprising administering to the patient a composition comprisingT-cell anergy suppressor, wherein the T-cell anergy suppressor reducesthe expression or activity of a T-cell anergy gene listed in Table 2. Insome additional aspects, this method is wherein the T-cell anergysuppressor is an antisense nucleic acid molecule targeted to a T-cellanergy gene listed in Table 2.

In further embodiments, there are methods for promoting a T-cellresponse in a subject comprising administering to the subject aneffective amount of a composition comprising T-cell anergy suppressor,wherein the T-cell anergy suppressor reduces the expression or activityof a T-cell anergy gene listed in Table 2. Such a T-cell anergysuppressor may be a small molecule inhibitor, an antibody, or the otherlike therapeutic that can, for example, manipulate a particular geneproduct. In a related aspect, the method may further compriseadministering to the subject one or more antigens, and may or may not bewherein the one or more antigens is in the same composition as theT-cell anergy suppressor. In a further related aspect, the T-cell anergysuppressor reduces expression of a T-cell anergy gene (which may be, insome embodiments, wherein the T-cell anergy suppressor is an antisensenucleic acid molecule targeted to a T-cell anergy gene listed in Table 2and may or may not be wherein the composition comprises multipleantisense nucleic acid molecules targeted to the same or different geneslisted in Table 2).

In particular embodiments, the subject or patient is suspected of havingcancer and/or has symptoms of cancer. Alternatively, the patient may bedeemed to be at risk for cancer (patient history, family history, orother risk factor(s)), at risk for metastasis, or at risk forrecurrence. In other embodiments, the patient has been diagnosed withcancer or pre-cancer. In other embodiments, the subject or patient has adifferent disease or condition, but that may involve a T-cell response,in particular where promotion of a T cell response may have sometherapeutic benefit.

In some embodiments there is an array, microarray, or chip comprisingone or more nucleic acid probes for each of at least 5 T-cell anergicgenes in Table 2. In some embodiments, the array, microarray or chipcomprises one or more nucleic acid probes for each of at least 10, orfor each at least 20, T-cell anergic genes in Table 2.

In related embodiments, a kit is provided, the kit comprising one ormore nucleic acid probes for each of at least 5, or for each of at least10, T-cell anergic genes in Table 2 and one or more reagents fordetecting expression of the T-cell anergic gene. In related aspects, thekit may be wherein the nucleic acid probes are located on an array,microarray, or chip. Kits are also provided wherein protein orpolypeptide products of genes are targeted, such as by antibodies,immunohistochemistry, or flow cytometry. In certain embodiments, a kitcomprises one or more molecules that target at least 5 or at least 10products of the T-cell anergic genes in Table 2. Examples of suchmolecules that may be used to target products of T-cell anergic genesinclude, but are not limited to, antibodies, antibody fragments,aptamers, or small molecules.

Methods may further involve calculating a risk score for the biologicalsample that characterizes or qualifies the anergy level of the patient'sT-cells. The levels may be qualified into percentiles, such as quartilesor deciles.

The weight of a the expression level (or the value of that expressionlevel) of a particular T-cell anergy gene reflects its importance in theaccuracy, specificity, integrity, or other parameter relating toquality, of the test. This can be implemented in the algorithm orreflected in a model coefficient. A person of ordinary skill in the artwould know how to determine this based on the experimental data. Incertain embodiments, weighing a value more heavily may involve adding ormultiplying the value by a particular number such as 0.01, 0.02, 0.03,0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7,0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1,2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5,3.6, 3.7. 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9,5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3,6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7,7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1,9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10.0, 10.5, 11.0, 11.5, 12.0,12.5, 13.0, 13.5, 14.0, 14.5, 15.0, 15.5, 16.0, 16.5, 17.0, 17.5, 18.0,18.5, 19.0. 19.5, 20.0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50,51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68,69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86,87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 105, 110, 115,120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185,190, 195, 200, 205, 210, 215, 220, 225, 230, 235, 240, 245, 250, 255,260, 265, 270, 275, 280, 285, 290, 295, 300, 305, 310, 315, 320, 325,330, 335, 340, 345, 350, 355, 360, 365, 370, 375, 380, 385, 390, 395,400, 410, 420, 425, 430, 440, 441, 450, 460, 470, 475, 480, 490, 500,510, 520, 525, 530, 540, 550, 560, 570, 575, 580, 590, 600, 610, 620,625, 630, 640, 650, 660, 670, 675, 680, 690, 700, 710, 720, 725, 730,740, 750, 760, 770, 775, 780, 790, 800, 810, 820, 825, 830, 840, 850,860, 870, 875, 880, 890, 900, 910, 920, 925, 930, 940, 950, 960, 970,975, 980, 990, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800,1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000,3100, 3200, 3300, 3400, 3500, 3600, 3700, 3800, 3900, 4000, 4100, 4200,4300, 4400, 4500, 4600, 4700, 4800, 4900, 5000, 6000, 7000, 8000, 9000,10000, or any range derivable therein.

In some embodiments, methods include evaluating a plurality ofexpression level values using a scoring algorithm to generate adiagnostic or risk score for having (or not having) anergic T-cells,wherein the patient is identified as having or as not having such abased on the score. It is understood by those of skill in the art thatthe score is a predictive value about whether the patient does or doesnot have T cells that are anergic. In some embodiments, the absence orpaucity of infiltrating T cells in a tumor and the level of expressionof one or more T-cell anergy genes may be a characteristic consistentwith T-cell anergy. In some embodiments, a report is generated and/orprovided that identifies the diagnostic score or the values that factorinto such a score. In some embodiments, a cut-off score is employed tocharacterize a sample as likely having anerigc T-cells (or alternativelynot having anergic T-cells). In some embodiments, the risk score for thepatient is compared to a cut-off score to characterize the biologicalsample from the patient with respect to whether they are likely torespond to immunotherapy.

In certain embodiments, T-cell anergy may be characterized as havingincreased expression of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50genes in Table 2 (or any range derivable therein) have at least about orat most about a two-, three-, four-, five-, 6-, 7-, 8-, 9-, 10-, 12-,15-, 20-, 25-, 30-, 35-, 40-, 45-, 50-, 60-, 70-, 80-, 90-, 100-foldincrease in expression (or any range derivable therein) as compared to areference or control level. In a general sense, “anergy” refers to theinability of an immune cell to mount a complete response against itstarget.

In some embodiments, a tangible computer-readable medium is provided,the tangible computer-readable medium comprising computer-readable codethat, when executed by a computer, causes the computer to performoperations comprising: a) receiving information corresponding to a levelof gene expression in a T-cell sample from a patient comprising one ormore T-cell anergy genes in Table 2; and b) calculating a risk score forthe sample that identifies the sample as containing anergic T-cells. Infurther embodiments, a tangible computer-readable medium is provided,the a tangible computer-readable medium comprising computer-readablecode that, when executed by a computer, causes the computer to performoperations comprising: a) receiving information corresponding to a levelof gene expression in a T-cell sample from a patient comprising one ormore T-cell anergy genes in Table 2; and b) calculating a risk score forthe sample that identifies the sample as containing T-cells that are notanergic. In related aspects, any of the tangible computer-readable mediamay be wherein calculating a risk score comprises using a computer andan algorithm.

In some embodiments, a method that comprises administration of antibodytherapy for treating a subject with an anergic T-cell associateddisease, condition or pathology, is provided. In certain aspects, theanergic T-cell associated disease is cancer. In other aspects, theanergic T-cell associated disease, condition or pathology is metaplasia,dysplasia, hyperplasia or neoplasia. In certain aspects neoplasia can beclassified as benign, pre-malignant or malignant.

In some embodiments a method of treating a subject with an anergicT-cell associated disease, condition or pathology, comprisesadministering a binding polypeptide that specifically recognizes andbinds a substance, such as another polypeptide. In certain embodiments,the binding polypeptide is an antibody or a binding fragment of anantibody. In particular embodiments, the binding polypeptide comprisesat least 1, 2, 3, 4, 5, or 6 CDRs from a monoclonal antibody. In certainembodiments, the method comprises an antibody therapy that comprisesmonoclonal antibodies. In yet other aspects the antibody therapycomprises isolated monoclonal antibodies. In some embodiments anantibody that is part of the antibody therapy binds a T-cell anergy geneproduct of at least one of the T-cell anergy genes listed in Table 2. Inyet other embodiments the antibody binds a cell-surface receptor that isthe product of at least one of the T-cell anergy genes listed in Table2. In still other embodiments, the antibody therapy comprises at leastone antibody that binds Semaphorin 7A (Sema7A), Class-I-MHC-restricted Tcell associated molecule (Crtam), lymphocyte-activation gene 3 (Lag3),tumor necrosis factor receptor superfamily member 9 (Tnfrsf9, also knownas 4-1BB), Neuritin (Nrn1), CLIP (CD74), Tnfsf11 (RANKL, CD254),prostaglandin F2 receptor inhibitor (Ptgfrn, CD9-P1, CD315) or oxidizedlow density lipoprotein receptor 1 (LOX1, OLR1). In specificembodiments, the antibody therapy comprises at least two antibodies thatbind different cell surface receptors selected from the T-cell anergygenes of Table 2. In additional embodiments, the antibody therapycomprises at least three, four or five antibodies that bind differentcell surface receptors selected from the T-cell anergy genes of Table 2.In one specific embodiment, the antibody therapy comprises a monoclonalantibody that binds lymphocyte-activation gene 3 (Lag3). In certainembodiments the anti-Lag3 antibody is monoclonal clone C9B7W. In certainembodiments the anti-Lag-3 antibody is BMS-986016. In certainembodiments the anti-Lag-3 antibody is any antibody in Table 3A or Table3B or any antibody comprising the CDRs (e.g., a fully human, humanizedor chimeric antibody) of an anti-Lag3 antibody of Table 3A or Table 3B.In another specific embodiment, the antibody therapy comprises amonoclonal antibody that binds 4-1BB (also known as tumor necrosisfactor receptor superfamily member 9 (Tnfrsf9)). In yet other aspects,the anti-4-1BB antibody is monoclonal antibody clone LOB12.3. In yetother aspects, the anti-4-1BB antibody is monoclonal antibodyBMS-663513, also known as urelumab. In certain embodiments theanti-anti-4-1BB antibody is any antibody in Table 4A or Table 4B or anyantibody comprising the CDRs (e.g., a fully human, humanized or chimericantibody) of an anti-4-1BB antibody of Table 4A or Table 4B. In stillother embodiments, the antibody therapy comprises a monoclonal antibodythat binds Lag3 and a monoclonal antibody that binds 4-1BB. In yet otherembodiments, antibody therapy may be administered in combination withother treatments contemplated herein to suppress the activity orexpression of T-cell anergy genes or their products.

In certain embodiments there is a method for treating a patientcomprising administering to the patient a composition comprising aT-cell anergy suppressor, wherein the T-cell anergy suppressor is anantibody, binding fragment thereor, or binding polypeptide. In certainaspects, the antibody is a monoclonal antibody. In yet other aspects thecomposition comprises isolated monoclonal antibodies. In someembodiments an antibody that is present in the composition binds aT-cell anergy gene product of at least one of the T-cell anergy geneslisted in Table 2. In yet other embodiments the antibody binds acell-surface receptor that is the product of at least one of the T-cellanergy genes listed in Table 2. In still other embodiments, thecomposition comprises at least one antibody that binds Semaphorin 7A(Sema7A), Class-I-MHC-restricted T cell associated molecule (Crtam),lymphocyte-activation gene 3 (Lag3), tumor necrosis factor receptorsuperfamily member 9 (Tnfrsf9, also known as 4-1BB), Neuritin (Nrn1),CLIP (CD74), Tnfsf11 (RANKL, CD254), prostaglandin F2 receptor inhibitor(Ptgfrn, CD9-P1, CD315) or oxidized low density lipoprotein receptor 1(LOX1, OLR1). In specific embodiments, the composition comprises atleast two antibodies that bind different cell surface receptors selectedfrom the T-cell anergy genes of Table 2. In additional embodiments, thecomposition comprises at least three, four or five antibodies that binddifferent cell surface receptors selected from the T-cell anergy genesof Table 2. In one specific embodiment, the composition comprises amonoclonal antibody that binds lymphocyte-activation gene 3 (Lag3). Incertain embodiments the anti-Lag3 antibody is monoclonal clone C9B7W. Incertain embodiments the anti-Lag-3 antibody is BMS-986016. In certainembodiments the anti-Lag-3 antibody is any antibody in Table 3A or Table3B or any antibody comprising the CDRs (e.g., a fully human, humanizedor chimeric antibody) of an anti-Lag3 antibody of Table 3A or Table 3B.In another specific embodiment, the composition comprises a monoclonalantibody that binds 4-1BB (also known as tumor necrosis factor receptorsuperfamily member 9 (Tnfrsf9)). In yet other aspects, the anti-4-1BBantibody is monoclonal antibody clone LOB12.3. In yet other aspects, theanti-4-1BB antibody is monoclonal antibody BMS-663513, also known asurelumab. In certain embodiments the anti-anti-4-1BB antibody is anyantibody in Table 4A or Table 4B or any antibody comprising the CDRs(e.g., a fully human, humanized or chimeric antibody) of an anti-4-1BBantibody of Table 4A or Table 4B. In still other embodiments, thecomposition comprises a monoclonal antibody that binds Lag3 and amonoclonal antibody that binds 4-1BB. In yet other embodiments, thecomposition may be administered in combination with other treatmentscontemplated herein to suppress the activity or expression of T-cellanergy genes or their products.

The T-cell anergy suppressor may be any polypeptide that specificallybinds T-cell anergy gene product from Table 2 or a T-cell anergy relatedcell surface receptor from Table 2 that includes Semaphorin 7A (Sema7A),Class-I-MHC-restricted T cell associated molecule (Crtam),lymphocyte-activation gene 3 (Lag3), tumor necrosis factor receptorsuperfamily member 9 (Tnfrsf9, also known as 4-1BB), Neuritin (Nrn1),CLIP (CD74), Tnfsf11 (RANKL, CD254), prostaglandin F2 receptor inhibitor(Ptgfrn, CD9-P1, CD315) or oxidized low density lipoprotein receptor 1(LOX1, OLR1). In certain embodiments, the binding polypeptide is apurified monoclonal antibody or a purified polyclonal antibody. Thepolypeptide may be, for example, an antibody that is single domain,humanized, or chimeric. In some embodiments, two or more bindingpolypeptides (e.g., two or more purified monoclonal antibodies orpurified polyclonal antibodies) may be administered to the patient. Incertain aspects, the binding polypeptide is recombinant. In otherembodiments, there may be chemical modifications to the polypeptide,such as the addition of one or more chemical modifications or moieties.

Embodiments are provided in which the binding polypeptide comprises oneor more CDR domains from an antibody that specifically binds to a T-cellanergy gene product from Table 2 or a T-cell anergy related cell surfacereceptor from Table 2 that includes Semaphorin 7A (Sema7A),Class-I-MHC-restricted T cell associated molecule (Crtam),lymphocyte-activation gene 3 (Lag3), tumor necrosis factor receptorsuperfamily member 9 (Tnfrsf9, also known as 4-1BB), Neuritin (Nrn1),CLIP (CD74), Tnfsf11 (RANKL, CD254), prostaglandin F2 receptor inhibitor(Ptgfrn, CD9-P1, CD315) or oxidized low density lipoprotein receptor 1(LOX1, OLR1). In particular embodiments, the binding polypeptidecomprises one, two, three, four, five, six, or more CDR domains fromamong the VH or VL domain of the monoclonal antibodies listed in Table3A, 3B, 4A or 4B. In certain aspects, the binding polypeptide comprisessix CDR domains from among the VH or VL domains of the monoclonalantibodies listed in Table 3A, 3B, 4A or 4B. In some embodiments, thebinding polypeptide comprises a sequence at least or at most 70%, 75%,80%, 85%, 90%, 95%, or 99% (or any range derivable therein) identical tothe VH or VL domain of the monoclonal antibodies listed in Table 3A, 3B,4A or 4B. Embodiments are provided in which the binding polypeptidecomprises the VH domain from the monoclonal antibodies listed in Table3A, 3B, 4A or 4B and/or the VL domain of the monoclonal antibodieslisted in Table 3A, 3B, 4A or 4B. In further embodiments, the monoclonalantibody is one or more monoclonal antibodies listed in Table 3A, 3B, 4Aor 4B.

In some embodiments the binding polypeptide comprises one or more CDRdomains from a binding polypeptide that specifically binds to a T-cellanergy gene product from Table 2 or a T-cell anergy related cell surfacereceptor from Table 2 that includes Semaphorin 7A (Sema7A),Class-I-MHC-restricted T cell associated molecule (Crtam),lymphocyte-activation gene 3 (Lag3), tumor necrosis factor receptorsuperfamily member 9 (Tnfrsf9, also known as 4-1BB), Neuritin (Nrn1),CLIP (CD74), Tnfsf11 (RANKL, CD254), prostaglandin F2 receptor inhibitor(Ptgfrn, CD9-P1, CD315) or oxidized low density lipoprotein receptor 1(LOX1, OLR1) and a scaffold from a polypeptide selected from the groupconsisting of an immunoglobulin, a fibronectin or a S. aureus protein Z.

The binding polypeptide may be operatively coupled to a second bindingpolypeptide. In some aspects, the first and second binding peptides areoperatively coupled recombinantly. In other aspects, the first andsecond binding peptides are operatively coupled chemically.

Embodiments are provided in which the binding polypeptide isadministered at a dose of about, at least about, or at most about 0.1mg/kg to 5 mg/kg, 1 mg/kg to 5 mg/kg, 0.1 mg/kg to 1 mg/kg, or 2 mg/kgto 5 mg/kg (or any range derivable therein).

Embodiments also provide a purified polypeptide comprising one or morebinding polypeptide CDR domains from an antibody that specifically bindsto a T-cell anergy gene product from Table 2 or a T-cell anergy relatedcell surface receptor from Table 2 that includes Semaphorin 7A (Sema7A),Class-I-MHC-restricted T cell associated molecule (Crtam),lymphocyte-activation gene 3 (Lag3), tumor necrosis factor receptorsuperfamily member 9 (Tnfrsf9, also known as 4-1BB), Neuritin (Nrn1),CLIP (CD74), Tnfsf11 (RANKL, CD254), prostaglandin F2 receptor inhibitor(Ptgfrn, CD9-P1, CD315) or oxidized low density lipoprotein receptor 1(LOX1, OLR1). In certain embodiments, the binding polypeptide competesfor binding of a a T-cell anergy gene product from Table 2 or a T-cellanergy related cell surface receptor from Table 2 that includesSemaphorin 7A (Sema7A), Class-I-MHC-restricted T cell associatedmolecule (Crtam), lymphocyte-activation gene 3 (Lag3), tumor necrosisfactor receptor superfamily member 9 (Tnfrsf9, also known as 4-1BB),Neuritin (Nrn1), CLIP (CD74), Tnfsf11 (RANKL, CD254), prostaglandin F2receptor inhibitor (Ptgfrn, CD9-P1, CD315) or oxidized low densitylipoprotein receptor 1 (LOX1, OLR1) with the monoclonal antibodieslisted in Table 3A, 3B, 4A or 4B. In certain aspects, the polypeptidehas an association constant for a T-cell anergy gene product from Table2 or a T-cell anergy related cell surface receptor from Table 2 thatincludes Semaphorin 7A (Sema7A), Class-I-MHC-restricted T cellassociated molecule (Crtam), lymphocyte-activation gene 3 (Lag3), tumornecrosis factor receptor superfamily member 9 (Tnfrsf9, also known as4-1BB), Neuritin (Nrn1), CLIP (CD74), Tnfsf11 (RANKL, CD254),prostaglandin F2 receptor inhibitor (Ptgfrn, CD9-P1, CD315) or oxidizedlow density lipoprotein receptor 1 (LOX1, OLR1) of between about 0.1 and20 nM⁻¹, 0.5 and 10 nM⁻¹, or 1.0 and 10 nM⁻¹ as measured by ELISA. Thepolypeptide may comprise, for example, a single domain antibody bindingpolypeptide, a humanized antibody, or a chimeric antibody.

In certain embodiments, the polypeptide is recombinant. In certainaspects, the recombinant polypeptide comprises at least 90%, 95%, or 99%of one or more CDR domains from the VH or VL domain of the monoclonalantibodies listed in Table 3A, 3B, 4A or 4B. In some embodiments, therecombinant polypeptide comprises two, three, four, five, six, or moreCDR domains from the VH or VL domain of the monoclonal antibodies listedin Table 3A, 3B, 4A or 4B.

In some embodiments, a recombinant polypeptide comprises i) CDR1, CDR2,and/or CDR3 from the variable light chain of monoclonal antibodieslisted in Table 3A, 3B, 4A or 4B; and/or ii) CDR1, CDR2, and/or CDR3from the variable heavy chain of monoclonal antibodies listed in Table3A, 3B, 4A or 4B. The sequences for these CDRs can be found in Table 3Band 4B.

In some embodiments, there is a purified polypeptide comprising one ormore binding polypeptide CDR domains from an antibody that specificallybinds to a T-cell anergy gene product from Table 2 or a T-cell anergyrelated cell surface receptor from Table 2 that includes Semaphorin 7A(Sema7A), Class-I-MHC-restricted T cell associated molecule (Crtam),lymphocyte-activation gene 3 (Lag3), tumor necrosis factor receptorsuperfamily member 9 (Tnfrsf9, also known as 4-1BB), Neuritin (Nrn1),CLIP (CD74), Tnfsf11 (RANKL, CD254), prostaglandin F2 receptor inhibitor(Ptgfrn, CD9-P1, CD315) or oxidized low density lipoprotein receptor 1(LOX1, OLR1). As indicated above, the polypeptide may comprise 1, 2, 3,4, 5, or 6 CDRs from the light and/or heavy chain variable regions of anantibody. Table 3B and 4B provide different T-cell anergy related cellsurface receptor antibodies and their CDR1, CDR2, and CDR3 sequencesfrom both the light and heavy chain variable regions. In certainembodiments, a polypeptide contains CDR1, CDR2, and/or CDR3 from thelight chain variable region of a particular antibody. It is contemplatedthat while in some embodiments a polypeptide has a CDR1, CDR2, and CDR3from the variable region of a light chain and/or the variable region ofa heavy chain that the CDR1, CDR2, and CDR3 need not be from the sameantibody. While some polypeptides have CDR1, CDR2, and CDR3 from thesame antibody or based on the same antibody, given the overlap in aminoacid sequences, a CDR1 from one antibody may be substituted with a CDRfrom or based on another antibody. It is generally contemplated,however, that when a single set of CDR1, CDR2, and CDR3 are employedtogether that they all be from a light chain variable region or from aheavy chain variable region, but not a mix from both.

Alternatively, the polypeptide may contain a CDR1 sequence that is, isat most or is at least 70, 75, 80, 85, 90, 95, 96, 97, 98, 99, 100%identical (or any range derivable therein) to the entire sequence setforth in SEQ ID NOs:63, 64, 65, 66, 67, 68, 100, or 110 which are CDR1sequences from the light chain variable region of a T-cell anergyrelated cell surface receptor antibodies. Alternatively or additionally,the polypeptide may contain a CDR2 sequence that is, is at most or is atleast 70, 75, 80, 85, 90, 95, 96, 97, 98, 99, 100% identical (or anyrange derivable therein) to the entire sequence set forth in SEQ ID NOs:69, 70, 71, 72, 73, 74, 101, 111, which are CDR2 sequences from thelight chain variable region of T-cell anergy related cell surfacereceptor antibodies. Alternatively or additionally, the polypeptide maycontain a CDR3 sequence that is, is at most or is at least 70, 75, 80,85, 90, 95, 96, 97, 98, 99, 100% identical (or any range derivabletherein) to the entire sequence set forth in SEQ ID NOs:75, 76, 77, 78,79, 80, 102, or 112 which are CDR3 sequences from the light chainvariable region of a T-cell anergy related cell surface receptorantibodies. Alternatively or additionally, the polypeptide may contain aCDR1 sequence that is, is at most or is at least 70, 75, 80, 85, 90, 95,96, 97, 98, 99, 100% identical (or any range derivable therein) to theentire sequence set forth in SEQ ID NOs: 45, 46, 47, 48, 49, 50, 95, or105 which are CDR1 sequences from the heavy chain variable region of aT-cell anergy related cell surface receptor antibodies. Alternatively oradditionally, the polypeptide may contain a CDR2 sequence that is, is atmost or is at least 70, 75, 80, 85, 90, 95, 96, 97, 98, 99, 100%identical (or any range derivable therein) to the entire sequence setforth in SEQ ID NOs: 51, 52, 53, 54, 55, 56, 96, or 106, which are CDR2sequences from the heavy chain variable region of a T-cell anergyrelated cell surface receptor antibodies. Alternatively or additionally,the polypeptide may contain a CDR3 sequence that is, is at most or is atleast 70, 75, 80, 85, 90, 95, 96, 97, 98, 99, 100% identical (or anyrange derivable therein) to the entire sequence set forth in SEQ ID NOs:57, 58, 59, 60, 61, 62, 97 or 107 which are CDR3 sequences from theheavy chain variable region of a T-cell anergy related cell surfacereceptor antibodies.

Other embodiments provide a recombinant polypeptide that comprises oneor more CDR domain from an antibody that specifically binds to a T-cellanergy gene product from Table 2 or a T-cell anergy related cell surfacereceptor from Table 2 that includes Semaphorin 7A (Sema7A),Class-I-MHC-restricted T cell associated molecule (Crtam),lymphocyte-activation gene 3 (Lag3), tumor necrosis factor receptorsuperfamily member 9 (Tnfrsf9, also known as 4-1BB), Neuritin (Nrn1),CLIP (CD74), Tnfsf11 (RANKL, CD254), prostaglandin F2 receptor inhibitor(Ptgfrn, CD9-P1, CD315) or oxidized low density lipoprotein receptor 1(LOX1, OLR1) and a scaffold from a polypeptide selected from the groupconsisting of an immunoglobulin, a fibronectin or a S. aureus protein Z.If it further contemplated that any polypeptide may be attached, fusedor conjugated to an agent or substance, such a therapeutic moiety or adetectable moirty.

In other embodiments, the binding polypeptide is an antibody comprising(a) a heavy chain comprising said VH region, and a human hinge, CH1,CH2, and CH3 regions from an IgG1, IgG2, IgG3 or IgG4 subtype; and (b) alight chain comprising said VL region, and either a human kappa CL orhuman lambda CL.

Certain embodiments provide a purified monoclonal antibody thatspecifically binds to a a T-cell anergy gene product from Table 2 or aT-cell anergy related cell surface receptor from Table 2 that includesSemaphorin 7A (Sema7A), Class-I-MHC-restricted T cell associatedmolecule (Crtam), lymphocyte-activation gene 3 (Lag3), tumor necrosisfactor receptor superfamily member 9 (Tnfrsf9, also known as 4-1BB),Neuritin (Nrn1), CLIP (CD74), Tnfsf11 (RANKL, CD254), prostaglandin F2receptor inhibitor (Ptgfrn, CD9-P1, CD315) or oxidized low densitylipoprotein receptor 1 (LOX1, OLR1), wherein the purified monoclonalantibody is any monoclonal antibody listed in Table 3A, 3B, 4A or 4B.

In some aspects, the purified polypeptide does not consist of monoclonalantibodies listed in Table 3A, 3B, 4A or 4B. In other embodiments thepurified polypeptide is not an isolated mouse monoclonal antibody.

Other embodiments provide a pharmaceutical composition comprising one ormore purified binding polypeptides. In some embodiments, thepharmaceutical composition provides a single unit dose of the purifiedpolypeptide in a sealed container. The pharmaceutical composition maycomprise at least a second anti-cancer agent including, but not limitedto, a chemotherapeutic, a cancer vaccine composition or a polypeptidethat specifically binds to a second a T-cell anergy gene product fromTable 2 or a T-cell anergy related cell surface receptor from Table 2that includes Semaphorin 7A (Sema7A), Class-I-MHC-restricted T cellassociated molecule (Crtam), lymphocyte-activation gene 3 (Lag3), tumornecrosis factor receptor superfamily member 9 (Tnfrsf9, also known as4-1BB), Neuritin (Nrn1), CLIP (CD74), Tnfsf11 (RANKL, CD254),prostaglandin F2 receptor inhibitor (Ptgfrn, CD9-P1, CD315) or oxidizedlow density lipoprotein receptor 1 (LOX1, OLR1).

Certain embodiments, provide a polynucleotide comprising a nucleic acidsequence encoding a binding polypeptide.

Other embodiments provide an expression vector comprising a nucleic acidsequence encoding a binding polypeptide operably linked to an expressioncontrol sequence. Some embodiments provide a host cell comprising theexpression vector.

Embodiments also provide a method manufacturing a binding polypeptidecomprising expressing a nucleic acid sequence encoding the polypeptideoperably linked to an expression control sequence in a host cell.

Embodiments also provide for the use of antibodies in methods andcompositions for the treatment of T-cell anergy related disease,pathology, condition, tumor or cancer. In certain embodiments,compositions are used in the manufacture of medicaments for thetherapeutic and/or prophylactic treatment of T-cell anergy relateddisease, pathology, condition, tumor or cancer. Furthermore, in someembodiments there are methods and compositions that can be used to treator prevent T-cell anergy related disease, pathology, condition, tumor orcancer.

Certain aspects are directed to methods of reducing, treating orameliorating a T-cell anergy related disease, pathology, condition,tumor or cancer comprising administering to a patient having orsuspected of having a T-cell anergy related disease, pathology,condition, tumor or cancer an effective amount of one or more purifiedantibodies that specifically bind a a T-cell anergy gene product fromTable 2 or a T-cell anergy related cell surface receptor from Table 2that includes Semaphorin 7A (Sema7A), Class-I-MHC-restricted T cellassociated molecule (Crtam), lymphocyte-activation gene 3 (Lag3), tumornecrosis factor receptor superfamily member 9 (Tnfrsf9, also known as4-1BB), Neuritin (Nrn1), CLIP (CD74), Tnfsf11 (RANKL, CD254),prostaglandin F2 receptor inhibitor (Ptgfrn, CD9-P1, CD315) or oxidizedlow density lipoprotein receptor 1 (LOX1, OLR1). The antibody can be apurified polyclonal antibody, a purified monoclonal antibody, arecombinant polypeptide, or a fragment thereof. In certain aspects theantibody is humanized or human. In still further aspects the antibody isa recombinant antibody segment. In certain aspects a monoclonal antibodyincludes one or more of the monoclonal antibodies listed in Table 3A,3B, 4A or 4B. An antibody can be administered at a dose of 0.1, 0.5, 1,5, 10, 50, 100 mg or μg/kg to 5, 10, 50, 100, 500 mg or μg/kg, or anyrange derivable therein. The recombinant antibody segment can beoperatively coupled to a second recombinant antibody segment. In certainaspects the second recombinant antibody segment binds a second a T-cellanergy gene product from Table 2 or a T-cell anergy related cell surfacereceptor from Table 2 that includes Semaphorin 7A (Sema7A),Class-I-MHC-restricted T cell associated molecule (Crtam),lymphocyte-activation gene 3 (Lag3), tumor necrosis factor receptorsuperfamily member 9 (Tnfrsf9, also known as 4-1BB), Neuritin (Nrn1),CLIP (CD74), Tnfsf11 (RANKL, CD254), prostaglandin F2 receptor inhibitor(Ptgfrn, CD9-P1, CD315) or oxidized low density lipoprotein receptor 1(LOX1, OLR1. The method can further comprise administering a secondantibody that binds a second a T-cell anergy gene product from Table 2or a T-cell anergy related cell surface receptor from Table 2 thatincludes Semaphorin 7A (Sema7A), Class-I-MHC-restricted T cellassociated molecule (Crtam), lymphocyte-activation gene 3 (Lag3), tumornecrosis factor receptor superfamily member 9 (Tnfrsf9, also known as4-1BB), Neuritin (Nrn1), CLIP (CD74), Tnfsf11 (RANKL, CD254),prostaglandin F2 receptor inhibitor (Ptgfrn, CD9-P1, CD315) or oxidizedlow density lipoprotein receptor 1 (LOX1, OLR1). In certain aspects themethod further comprises administering anti-cancer compound orcomposition.

Embodiments are directed to monoclonal antibody polypeptides,polypeptides having one or more segments thereof, and polynucleotidesencoding the same. In certain aspects a polypeptide can comprise all orpart of the heavy chain variable region and/or the light chain variableregion of T-cell anergy related gene product antibodies. In a furtheraspect, a polypeptide can comprise an amino acid sequence thatcorresponds to a first, second, and/or third complementary determiningregions (CDRs) from the light variable chain and/or heavy variable chainof a T-cell anergy related gene product antibody.

In still further aspects, embodiments provide a hybridoma cell line thatproduces a monoclonal antibody of the embodiments. In embodiments thehybridoma cell line is a line that produces the monoclonal antibodieslisted in Table 3A, 3B, 4A or 4B. In a further aspect, 1, 2, and/or 3CDRs from the light and/or heavy chain variable region of a MAb can becomprised in a humanized antibody or variant thereof.

Certain aspects are directed to methods of treating a subject having orsuspected of having a T-cell anergy related disease, pathology,condition, tumor or cancer comprising administering to a patient havingor suspected of having a T-cell anergy related disease, pathology,condition, tumor or cancer an effective amount of a purified antibody orpolypeptide that specifically binds a a T-cell anergy gene product fromTable 2 or a T-cell anergy related cell surface receptor from Table 2that includes Semaphorin 7A (Sema7A), Class-I-MHC-restricted T cellassociated molecule (Crtam), lymphocyte-activation gene 3 (Lag3), tumornecrosis factor receptor superfamily member 9 (Tnfrsf9, also known as4-1BB), Neuritin (Nrn1), CLIP (CD74), Tnfsf11 (RANKL, CD254),prostaglandin F2 receptor inhibitor (Ptgfrn, CD9-P1, CD315) or oxidizedlow density lipoprotein receptor 1 (LOX1, OLR1).

In a further aspect methods are directed to treating a subject at riskof a T-cell anergy related disease, pathology, condition, tumor orcancer comprising administering to a patient at risk of a T-cell anergyrelated disease, pathology, condition, tumor or cancer an effectiveamount of an antibody that binds a a T-cell anergy gene product fromTable 2 or a T-cell anergy related cell surface receptor from Table 2that includes Semaphorin 7A (Sema7A), Class-I-MHC-restricted T cellassociated molecule (Crtam), lymphocyte-activation gene 3 (Lag3), tumornecrosis factor receptor superfamily member 9 (Tnfrsf9, also known as4-1BB), Neuritin (Nrn1), CLIP (CD74), Tnfsf11 (RANKL, CD254),prostaglandin F2 receptor inhibitor (Ptgfrn, CD9-P1, CD315) or oxidizedlow density lipoprotein receptor 1 (LOX1, OLR1) prior to development ofa T-cell anergy related disease, pathology, condition, tumor or cancer.

Certain embodiments are directed to an antibody or binding polypeptidecomposition comprising an isolated and/or recombinant antibody orpolypeptide that specifically binds a peptide segment as describedabove. In certain aspects the antibody or polypeptide has a sequencethat is, is at least, or is at most 80, 85, 90, 95, 96, 97, 98, 99, or100% identical (or any range derivable therein) to all or part of anymonoclonal antibody provided herein.

In some embodiments, a method for treating a patient with immunotherapyis provided, the method comprising administering immunotherapy to thepatient after the patient is identified as having anergic T-cells. Insome aspects, this method may be wherein: the patient is identified ashaving anergic T-cells by measuring increased expression level(s) of oneor more T-cell anergic genes in Table 2 compared to the expression levelto a reference or control level of expression in non-anergic T-cells; orthe patient is identified as having non-anergic T-cells by measuringexpression level(s) of one or more T-cell anergic genes in Table 2 andcomparing the expression level to a reference or control level ofexpression in anergic T-cells. In related aspects, an above method maybe wherein: the immunotherapy comprises a cell-based immunotherapy; theimmunotherapy may comprise antibody therapy, a vaccine (such as a cancervaccine), a checkpoint inhibitor, cytokine IL-2, or an adoptive T-celltherapy.

As used herein the specification, “a” or “an” may mean one or more. Asused herein in the claim(s), when used in conjunction with the word“comprising”, the words “a” or “an” may mean one or more than one.

The use of the term “or” in the claims is used to mean “and/or” unlessexplicitly indicated to refer to alternatives only or the alternativesare mutually exclusive, although the disclosure supports a definitionthat refers to only alternatives and “and/or.” As used herein “another”may mean at least a second or more.

Throughout this application, the term “about” is used to indicate that avalue includes the inherent variation of error for the device, themethod being employed to determine the value, or the variation thatexists among the study subjects.

Other objects, features and advantages of the present invention willbecome apparent from the following detailed description. It should beunderstood, however, that the detailed description and the specificexamples, while indicating preferred embodiments of the invention, aregiven by way of illustration only, since various changes andmodifications within the spirit and scope of the invention will becomeapparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and areincluded to further demonstrate certain aspects of the presentinvention. The invention may be better understood by reference to one ormore of these drawings in combination with the detailed description ofspecific embodiments presented herein.

FIG. 1A-1C—Identification of direct targets of Egr2 in the context of Tcell anergy by ChIP-seq and gene expression profiling analyses. CARTg×Egr2^(flox/flox) Th1 T cells were anergized with immobilizedanti-CD3, and Egr2-associated genes were identified by ChIP-seqanalysis. (A) Distribution of the types of Egr2 binding sites in thegenome. (B) Consensus sequence of Egr2 binding sites derived fromChIP-seq were highly similar to that published on TRANSFAC. EV- orCre-transduced CAR Tg×Egr2^(flox/flox) Th1 T cells were left untreatedor anergized by immobilized anti-CD3 mAb, and anergy-associated andEgr2-dependent genes were determined by gene chip analysis. The resultsfrom ChIP-seq and gene chip analyses were merged to identify the directtargets of Egr2. (C) A list of direct Egr2 targets and their foldchanges in expression upon anergy induction (Upregulation) and with Egr2deletion (Ratio EV vs. Cre). The results were summarized from twoindependent ChIP-seq analyses and three independent gene expressionprofiling analyses.

FIG. 2—Confirmatory ChIP assay on selected targets of Egr2. CARTg×Egr2^(flox/flox) Th1 T cells were left untreated (Control) oranergized with immobilized anti-CD3 mAb (Anergic), cross-linked, thecell lysate were immunoprecipitated by anti-Egr2-coated beads or emptybeads, and the association of Egr2 with the indicated genes wasdetermined by ChIP Assay. Data are presented as mean+/−SD, and arerepresentative of three independent experiments, **p<0.01.

FIG. 3—Confirmatory qRT-PCR on selected targets of Egr2. CARTg×Egr2^(flox/flox) Th1 clones were infected with an EV- or aCre-expressing adenovirus to delete Egr2. The cells were then leftuntreated (Control) or anergized with immobilized anti-CD3 mAb(Anergic), and the expression of the indicated genes was examined byqRT-PCR. Data are presented as mean+/−SD, and are representative ofthree independent experiments, * p<0.05, **p<0.01.

FIG. 4A-4D—Confirmatory protein expression on selected targets of Egr2.CAR Tg×Egr2^(flox/flox) Th1 clones were infected with an EV- or aCre-expressing adenovirus to delete Egr2. The cells were then leftuntreated (Control) or anergized with immobilized anti-CD3 mAb(Anergic), and the expressions of CCL1, Sema7A, Crtam, and Crabp2 wereexamined by ELISA (A), Immunoblot (B and D), and flow cytometry (C)respectively. Data are presented as mean+/−SD, and are representative oftwo to six independent experiments, **p<0.01.

FIG. 5—The role of Egr2 in regulating the indicated genes is confirmedin CAR Tg×Egr2^(flox/flox) Th1 T cell clone 11. (A-B) CARTg×Egr2^(flox/flox) Th1 clone 11 cells were infected with an EV- or aCre-expressing adenovirus to delete Egr2. The cells were then leftuntreated (Control) or anergized with immobilized anti-CD3 mAb(Anergic), and the expression of the indicated genes was examined byqRT-PCR. Data are presented as mean+/−SD, and are representative of twoindependent experiments, * p<0.05, **p<0.01.

FIG. 6—The role of Egr2 in regulating the indicated genes is confirmedin CAR Tg×Egr2^(flox/flox) Th1 T cell clone 46. (A-B) CARTg×Egr2^(flox/flox) Th1 clone 46 cells were infected with an EV- or aCre-expressing adenovirus to delete Egr2. The cells were then leftuntreated (Control) or anergized with immobilized anti-CD3 mAb(Anergic), and the expression of the indicated genes was examined byqRT-PCR. Data are presented as mean+/−SD, and are representative of twoindependent experiments, **p<0.01.

FIG. 7—Model for DGK-α in T cell anergy.

FIG. 8—DGK-α appears to be regulated by EGR2.

FIG. 9—Adeno-Cre transduction of CARTg×Egr2^(fl/fl) Th1 cells deletionof Egr2.

FIG. 10—Egr2 deletion leads to resistance to anergy induction in vitro.

FIG. 11—Egr2 directly regulates most of the known anergy associatedgenes: qRT-PCR.

FIG. 12—Model for Egr2 as central transcriptional regulator of T cellanergy.

FIG. 13—Strategy to determine global Egr2-driven transcriptional programin anergic T cells.

FIG. 14—46 genes identified as targets of Egr2 by gene array×ChIP-SEQ inanergy.

FIG. 15—New Egr2-dependent anergy associated genes.

FIG. 16—PD1, Lag3 and Crtam are highly upregulated on CD8+tumor-infiltrating lymphocytes (TILs) in the context of B16 melanoma.

FIG. 17—Lag3+Crtam+CD8+TILs are defective in IL-2 production upon exvivo stimulation.

FIG. 18—Lag3+Crtam+CD8+TILs are hypoproliferative upon ex vivostimulation.

FIG. 19—Anergy-associated genes are enriched in Lag3+Crtam+CD8+TILs.

FIG. 20—Conditional deletion of Eg2 in T cells leads to enhancedanti-tumor immune response and slowed tumor growth.

FIG. 21—B16.SIY tumor treatment with passive immunization of anti-4-1BBor anti-LAG-3 antibodies. Antibody treatments were on days 4, 7, 10 and13 post-tumor cell inoculation. Antibody dose was 100 μg of eachantibody, per mouse, per treatment.

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS I. T Cell Anergy

Certain embodiments are directed to methods relating to the diagnosis ordetermination of T-cell anergy or T-cells in a hyporesponsive state.Other embodiments are directed to methods relating to therapeutic,prognostic, and diagnostic applications surrounding T-cell anergy,anergic T-cells and T-cells in a hyporesponsive state, such as thosemethods and compositions related to cancer.

In some embodiments, methods are directed to detecting the expression ofgenes associated with T-cell anergy or T-cell hyporesponsiveness. Inother embodiments, methods are directed to the treatment or affectingthe expression of genes involved in T-cell anergy or T-cellhyporesponsiveness. In certain embodiments, methods of diagnosinginvolve measuring the protein or nucleic acid expression of the spectrumof genes/gene products upregulated in an Egr2-dependent fashion. Inother embodiments methods of treating involve affecting the expressionof the spectrum of genes/gene products upregulated in an Egr2-dependentfashion.

II. Nucleic Acids

Nucleic acid molecules identical or complementary to all or part of anyof the T cell anergy genes may be employed in diagnostic, prognostic andtherapeutic methods and compositions described herein.

In the disclosed methods, nucleic acids can be labeled, used as probes,in array analysis, or employed in other diagnostic or prognosticapplications, particularly those related to detecting T-cell anergy,T-cell pathological conditions and/or cancer. The expression of genesassociated with T-cell anergy, a T-cell anergic condition or T-cellhyporesponsiveness may be assayed or detected by methods used to detectand/or measure nucleic acid expression described below.

In addition nucleic acids can be used as antisense or siRNA moleculestargeted at a T cell anergy gene for use in reducing expression of thatgene. In certain embodiments, reduction of expression providesinhibition of T cell anergy, and accordingly, induction of a T cellresponse. In the context of cancer, T cell infiltration of a tumor hasbenefits, and T cell anergy is contraindicated with T cell infiltration.These therapeutic nucleic acids may be modified to enhance theirstability in storage or in vivo, bioavailability, activity, orlocalization.

The nucleic acids may have been endogenously produced by a cell, or beensynthesized or produced chemically or recombinantly. They may beisolated and/or purified. Nucleic acids used in methods and compositionsdisclosed herein may have regions of identity or complementarity toanother nucleic acid, such as a T cell anergy gene. It is contemplatedthat the region of complementarity or identity can be at least 5contiguous residues, though it is specifically contemplated that theregion is, is at least, or is at most 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50,51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68,69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86,87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 110, 120, 130,140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270,280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410,420, 430, 440, 441, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540,550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680,690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820,830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960,970, 980, 990, or 1000, or any range derivable therein, contiguousnucleotides. It is further understood that the length of complementaritywithin a gene, gene transcript or between a gene target and a nucleicacid are such lengths. Moreover, the complementarity may be expressed asa percentage, meaning that the complementarity between a probe and itstarget is 90% or greater over the length of the probe. In someembodiments, complementarity is or is at least 90%, 95% or 100%, or anyrange derivable therein. In particular, such lengths may be applied toany nucleic acid comprising a nucleic acid sequence identified. Thecommonly used name of the genes or gene targets is given throughout theapplication. Identification of gene sequence is provided by the Entrezgene number in the table in FIG. 1. This can be used to design thesequence of any probe, primer or siRNA molecule that is complementary oridentical to a target T cell anergy gene identified herein.

It is understood that a nucleic acid may be derived from genomicsequences or a gene. In this respect, the term “gene” is used forsimplicity to refer to the genomic sequence encoding the transcript fora given amino acid sequence. However, embodiments may involve genomicsequences of a gene that are involved in its expression, such as apromoter or other regulatory sequences.

The term “recombinant” generally refers to a molecule that has beenmanipulated in vitro or that is a replicated or expressed product ofsuch a molecule.

The term “nucleic acid” is well known in the art. A “nucleic acid” asused herein will generally refer to a molecule (one or more strands) ofDNA, RNA or a derivative or analog thereof, comprising a nucleobase. Anucleobase includes, for example, a naturally occurring purine orpyrimidine base found in DNA (e.g., an adenine “A,” a guanine “G,” athymine “T” or a cytosine “C”) or RNA (e.g., an A, a G, an uracil “U” ora C). The term “nucleic acid” encompasses the terms “oligonucleotide”and “polynucleotide,” each as a subgenus of the term “nucleic acid.”

As used herein, “hybridization”, “hybridizes” or “capable ofhybridizing” is understood to mean the forming of a double or triplestranded molecule or a molecule with partial double or triple strandednature. The term “anneal” is synonymous with “hybridize.” The term“hybridization”, “hybridize(s)” or “capable of hybridizing” encompassesthe terms “stringent condition(s)” or “high stringency” and the terms“low stringency” or “low stringency condition(s).”

As used herein, “stringent condition(s)” or “high stringency” are thoseconditions that allow hybridization between or within one or morenucleic acid strand(s) containing complementary sequence(s), butpreclude hybridization of random sequences. Stringent conditionstolerate little, if any, mismatch between a nucleic acid and a targetstrand. Such conditions are well known to those of ordinary skill in theart, and are preferred for applications requiring high selectivity.Non-limiting applications include isolating a nucleic acid, such as agene or a nucleic acid segment thereof, or detecting at least onespecific mRNA transcript or a nucleic acid segment thereof, and thelike.

Stringent conditions may comprise low salt and/or high temperatureconditions, such as provided by about 0.02 M to about 0.5 M NaCl attemperatures of about 42° C. to about 70° C. It is understood that thetemperature and ionic strength of a desired stringency are determined inpart by the length of the particular nucleic acid(s), the length andnucleobase content of the target sequence(s), the charge composition ofthe nucleic acid(s), and to the presence or concentration of formamide,tetramethylammonium chloride or other solvent(s) in a hybridizationmixture.

It is also understood that these ranges, compositions and conditions forhybridization are mentioned by way of non-limiting examples only, andthat the desired stringency for a particular hybridization reaction isoften determined empirically by comparison to one or more positive ornegative controls. Depending on the application envisioned it ispreferred to employ varying conditions of hybridization to achievevarying degrees of selectivity of a nucleic acid towards a targetsequence. In a non-limiting example, identification or isolation of arelated target nucleic acid that does not hybridize to a nucleic acidunder stringent conditions may be achieved by hybridization at lowtemperature and/or high ionic strength. Such conditions are termed “lowstringency” or “low stringency conditions,” and non-limiting examples ofsuch include hybridization performed at about 0.15 M to about 0.9 M NaClat a temperature range of about 20° C. to about 50° C. Of course, it iswithin the skill of one in the art to further modify the low or highstringency conditions to suite a particular application.

A nucleic acid may comprise, or be composed entirely of, a derivative oranalog of a nucleobase, a nucleobase linker moiety and/or backbonemoiety that may be present in a naturally occurring nucleic acid. RNAwith nucleic acid analogs may also be labeled according to methodsdisclosed herein. As used herein a “derivative” refers to a chemicallymodified or altered form of a naturally occurring molecule, while theterms “mimic” or “analog” refer to a molecule that may or may notstructurally resemble a naturally occurring molecule or moiety, butpossesses similar functions. As used herein, a “moiety” generally refersto a smaller chemical or molecular component of a larger chemical ormolecular structure. Nucleobase, nucleoside, and nucleotide analogs orderivatives are well known in the art, and have been described (see forexample, Scheit, 1980, incorporated herein by reference).

Additional non-limiting examples of nucleosides, nucleotides, or nucleicacids comprising 5-carbon sugar and/or backbone moiety derivatives oranalogs, include those in: U.S. Pat. No. 5,681,947, which describesoligonucleotides comprising purine derivatives that form triple helixeswith and/or prevent expression of dsDNA; U.S. Pat. Nos. 5,652,099 and5,763,167, which describe nucleic acids incorporating fluorescentanalogs of nucleosides found in DNA or RNA, particularly for use asfluorescent nucleic acid probes; U.S. Pat. No. 5,614,617, whichdescribes oligonucleotide analogs with substitutions on pyrimidine ringsthat possess enhanced nuclease stability; U.S. Pat. Nos. 5,670,663,5,872,232 and 5,859,221, which describe oligonucleotide analogs withmodified 5-carbon sugars (i.e., modified 2′-deoxyfuranosyl moieties)used in nucleic acid detection; U.S. Pat. No. 5,446,137, which describesoligonucleotides comprising at least one 5-carbon sugar moietysubstituted at the 4′ position with a substituent other than hydrogenthat can be used in hybridization assays; U.S. Pat. No. 5,886,165, whichdescribes oligonucleotides with both deoxyribonucleotides with 3′-5′internucleotide linkages and ribonucleotides with 2′-5′ internucleotidelinkages; U.S. Pat. No. 5,714,606, which describes a modifiedinternucleotide linkage wherein a 3′-position oxygen of theinternucleotide linkage is replaced by a carbon to enhance the nucleaseresistance of nucleic acids; U.S. Pat. No. 5,672,697, which describesoligonucleotides containing one or more 5′ methylene phosphonateinternucleotide linkages that enhance nuclease resistance; U.S. Pat.Nos. 5,466,786 and 5,792,847, which describe the linkage of asubstituent moiety which may comprise a drug or label to the 2′ carbonof an oligonucleotide to provide enhanced nuclease stability and abilityto deliver drugs or detection moieties; U.S. Pat. No. 5,223,618, whichdescribes oligonucleotide analogs with a 2 or 3 carbon backbone linkageattaching the 4′ position and 3′ position of adjacent 5-carbon sugarmoiety to enhanced cellular uptake, resistance to nucleases andhybridization to target RNA; U.S. Pat. No. 5,470,967, which describesoligonucleotides comprising at least one sulfamate or sulfamideinternucleotide linkage that are useful as nucleic acid hybridizationprobe; U.S. Pat. Nos. 5,378,825, 5,777,092, 5,623,070, 5,610,289 and5,602,240, which describe oligonucleotides with three or four atomlinker moiety replacing phosphodiester backbone moiety used for improvednuclease resistance, cellular uptake, and regulating RNA expression;U.S. Pat. No. 5,858,988, which describes hydrophobic carrier agentattached to the 2′-O position of oligonucleotides to enhanced theirmembrane permeability and stability; U.S. Pat. No. 5,214,136, whichdescribes oligonucleotides conjugated to anthraquinone at the 5′terminus that possess enhanced hybridization to DNA or RNA; enhancedstability to nucleases; U.S. Pat. No. 5,700,922, which describesPNA-DNA-PNA chimeras wherein the DNA comprises2′-deoxy-erythro-pentofuranosyl nucleotides for enhanced nucleaseresistance, binding affinity, and ability to activate RNase H; and U.S.Pat. No. 5,708,154, which describes RNA linked to a DNA to form aDNA-RNA hybrid; U.S. Pat. No. 5,728,525, which describes the labeling ofnucleoside analogs with a universal fluorescent label.

Additional teachings for nucleoside analogs and nucleic acid analogs areU.S. Pat. No. 5,728,525, which describes nucleoside analogs that areend-labeled; U.S. Pat. Nos. 5,637,683, 6,251,666 (L-nucleotidesubstitutions), and U.S. Pat. No. 5,480,980 (7-deaza-2′deoxyguanosinenucleotides and nucleic acid analogs thereof).

A. Modified Nucleotides

Labeling methods and kits may use nucleotides that are both modified forattachment of a label and can be incorporated into a nucleic acidmolecule. Such nucleotides include those that can be labeled with a dye,including a fluorescent dye, or with a molecule such as biotin. Labelednucleotides are readily available; they can be acquired commercially orthey can be synthesized by reactions known to those of skill in the art.

Modified nucleotides for use in the methods and compositions are notnaturally occurring nucleotides, but instead, refer to preparednucleotides that have a reactive moiety on them. Specific reactivefunctionalities of interest include: amino, sulfhydryl, sulfoxyl,aminosulfhydryl, azido, epoxide, isothiocyanate, isocyanate, anhydride,monochlorotriazine, dichlorotriazine, mono- or dihalogen substitutedpyridine, mono- or disubstituted diazine, maleimide, epoxide, aziridine,sulfonyl halide, acid halide, alkyl halide, aryl halide, alkylsulfonate,N-hydroxysuccinimide ester, imido ester, hydrazine, azidonitrophenyl,azide, 3-(2-pyridyl dithio)-propionamide, glyoxal, aldehyde, iodoacetyl,cyanomethyl ester, p-nitrophenyl ester, o-nitrophenyl ester,hydroxypyridine ester, carbonyl imidazole, and other such chemicalgroups. In some embodiments, the reactive functionality may be bondeddirectly to a nucleotide, or it may be bonded to the nucleotide througha linking group. The functional moiety and any linker cannotsubstantially impair the ability of the nucleotide to be added to thenucleic acid molecule or to be labeled. Representative linking groupsinclude carbon containing linking groups, typically ranging from about 2to 18, usually from about 2 to 8 carbon atoms, where the carboncontaining linking groups may or may not include one or moreheteroatoms, e.g. S, O, N etc., and may or may not include one or moresites of unsaturation. Of particular interest in some embodiments arealkyl linking groups, typically lower alkyl linking groups of 1 to 16,usually 1 to 4 carbon atoms, where the linking groups may include one ormore sites of unsaturation. The functionalized nucleotides (or primers)used in the above methods of functionalized target generation may befabricated using known protocols or purchased from commercial vendors,e.g., Sigma, Roche, Ambion, etc. Functional groups may be preparedaccording to ways known to those of skill in the art, including therepresentative information found in U.S. Pat. Nos. 4,404,289; 4,405,711;4,337,063 and 5,268,486, and U.K. Patent 1,529,202, which are allincorporated by reference.

Amine-modified nucleotides are used in some embodiments. Theamine-modified nucleotide is a nucleotide that has a reactive aminegroup for attachment of the label. It is contemplated that anyribonucleotide (G, A, U, or C) or deoxyribonucleotide (G, A, T, or C)can be modified for labeling. Examples include, but are not limited to,the following modified ribo- and deoxyribo-nucleotides:5-(3-aminoallyl)-UTP; 8-[(4-amino)butyl]-amino-ATP and8-[(6-amino)butyl]-amino-ATP; N6-(4-amino)butyl-ATP,N6-(6-amino)butyl-ATP, N4-[2,2-oxy-bis-(ethylamine)]-CTP;N6-(6-Amino)hexyl-ATP; 8-[(6-Amino)hexyl]-amino-ATP;5-propargylamino-CTP, 5-propargylamino-UTP; 5-(3-aminoallyl)-dUTP;8-[(4-amino)butyl]-amino-dATP and 8-[(6-amino)butyl]-amino-dATP;N6-(4-amino)butyl-dATP, N6-(6-amino)butyl-dATP,N4-[2,2-oxy-bis-(ethylamine)]-dCTP; N6-(6-Amino)hexyl-dATP;8-[(6-Amino)hexyl]-amino-dATP; 5-propargylamino-dCTP, and5-propargylamino-dUTP. Such nucleotides can be prepared according tomethods known to those of skill in the art. Moreover, a person ofordinary skill in the art could prepare other nucleotide entities withthe same amine-modification, such as a 5-(3-aminoallyl)-CTP, GTP, ATP,dCTP, dGTP, dTTP, or dUTP in place of a 5-(3-aminoallyl)-UTP.

Moreover, nucleic acid molecules may be modified through covalentbonding, attachment, fusion, or conjugation to or with one or morecompounds. Examples of modifications include those identified in U.S.Patent Publications 20050261218 and 20090286969, which are herebyincorporated by reference. One or more modifications may be at the 5′end, the 3′ end, on both, as well as on one or moth strands of adouble-stranded molecule. In some embodiments, the modification is onthe strand that is the antisense strand relative to the targetedtranscript, or it may be on the complementary strand to the antisensestrand. Modifications may be to the sugar molecule or to the backbone ofthe nucleic acid residue.

In some embodiments, nucleic acid molecules comprise at least 50%, atleast 60%, at least 70%, at least 75%, at least 80%, or at least 85%sequence complementarity to a target region within the target nucleicacid. In other embodiments, the molecules comprise at least 90% sequencecomplementarity to a target region within the target nucleic acid. Inother embodiments, the molecules comprise at least 95% or at least 99%sequence complementarity to a target region within the target nucleicacid. For example, a nucleic acid molecule in which 18 of 20 nucleobasesof it are complementary to a target sequence would represent 90 percentcomplementarity. In this example, the remaining noncomplementarynucleobases may be clustered or interspersed with complementarynucleobases and need not be contiguous to each other or to complementarynucleobases. As such, an oligomeric compound that is 18 nucleobases inlength having 4 noncomplementary nucleobases that are flanked by tworegions of complete complementarity with the target nucleic acid wouldhave 77.8% overall complementarity with the target nucleic acid. Percentcomplementarity of an oligomeric compound with a region of a targetnucleic acid can be determined routinely using BLAST programs (basiclocal alignment search tools) and PowerBLAST programs known in the art(Altschul et al., J. Mol. Biol., 1990, 215, 403-410; Zhang and Madden,Genome Res., 1997, 7, 649-656).

A nucleic acid molecule is “targeted” to a molecule (that is a nucleicacid) in some embodiments. This means there is sufficient sequencecomplementarity to achieve a level of hybridization that accomplishes aparticular goal with respect to the nucleic acid molecule, such as inthe context of being a probe, a primer or an siRNA. In some embodiments,expression or function is to be modulated. This targeted nucleic acid(or nucleic acid or gene target) may be, for example, a mRNA transcribedfrom a cellular gene whose expression is associated with a particulardisorder or disease state, a small non-coding RNA or its precursor, or anucleic acid molecule from an infectious agent.

The targeting process usually also includes determination of at leastone target region, segment, or site within the target nucleic acid forthe interaction to occur such that the desired effect, e.g., modulationof levels, expression or function, will result. Within the context ofthe present invention, the term “region” is defined as a portion of thetarget nucleic acid having at least one identifiable sequence,structure, function, or characteristic. Within regions of target nucleicacids are segments. “Segments” are defined as smaller or sub-portions ofregions within a target nucleic acid. “Sites,” as used in the presentinvention, are defined as specific positions within a target nucleicacid. The terms region, segment, and site can also be used to describean oligomeric compound of the invention such as for example a gappedoligomeric compound having three separate segments.

Targets of the nucleic acid molecules described in embodiments includeboth coding and non-coding nucleic acid sequences. For coding nucleicacid sequences, the translation initiation codon is typically 5′-AUG (intranscribed mRNA molecules; 5′-ATG in the corresponding DNA molecule),the translation initiation codon is also referred to as the “AUG codon,”the “start codon” or the “AUG start codon.” A minority of genes have atranslation initiation codon having the RNA sequence 5′-GUG, 5′-UUG or5′-CUG, and 5′-AUA, 5′-ACG and 5′-CUG have been shown to function invivo. Thus, the terms “translation initiation codon” and “start codon”can encompass many codon sequences, even though the initiator amino acidin each instance is typically methionine (in eukaryotes). It is alsoknown in the art that eukaryotic and prokaryotic genes may have two ormore alternative start codons, any one of which may be preferentiallyutilized for translation initiation in a particular cell type or tissue,or under a particular set of conditions. In the context of theinvention, “start codon” and “translation initiation codon” refer to thecodon or codons that are used in vivo to initiate translation of an mRNAtranscribed from a gene encoding a nucleic acid target, regardless ofthe sequence(s) of such codons. It is also known in the art that atranslation termination codon (or “stop codon”) of a gene may have oneof three sequences, i.e., 5′-UAA, 5′-UAG and 5′-UGA (the correspondingDNA sequences are 5′-TAA, 5′-TAG and 5′-TGA, respectively).

B. Preparation of Nucleic Acids

A nucleic acid may be made by any technique known to one of ordinaryskill in the art, such as for example, chemical synthesis, enzymaticproduction, or biological production. It is specifically contemplatedthat nucleic acid probes are chemically synthesized.

In some embodiments, nucleic acids are recovered or isolated from abiological sample. The nucleic acids may be recombinant or it may benatural or endogenous to the cell (produced from the cell's genome). Itis contemplated that a biological sample may be treated in a way so asto enhance the recovery of small RNA molecules such as mRNA or miRNAs.U.S. patent application Ser. No. 10/667,126 describes such methods andis specifically incorporated herein by reference. Generally, methodsinvolve lysing cells with a solution having guanidinium and a detergent.

Alternatively, nucleic acid synthesis is performed according to standardmethods. See, for example, Itakura and Riggs (1980). Additionally, U.S.Pat. Nos. 4,704,362, 5,221,619, and 5,583,013 each describe variousmethods of preparing synthetic nucleic acids. Non-limiting examples of asynthetic nucleic acid (e.g., a synthetic oligonucleotide) include anucleic acid made by in vitro chemical synthesis using phosphotriester,phosphite, or phosphoramidite chemistry and solid phase techniques suchas described in EP 266,032, incorporated herein by reference, or viadeoxynucleoside H-phosphonate intermediates as described by Froehler etal., 1986 and U.S. Pat. No. 5,705,629, each incorporated herein byreference. In some methods, one or more oligonucleotide may be used.Various different mechanisms of oligonucleotide synthesis have beendisclosed in for example, U.S. Pat. Nos. 4,659,774, 4,816,571,5,141,813, 5,264,566, 4,959,463, 5,428,148, 5,554,744, 5,574,146,5,602,244, each of which is incorporated herein by reference.

A non-limiting example of an enzymatically produced nucleic acid includeone produced by enzymes in amplification reactions such as PCR™ (see forexample, U.S. Pat. Nos. 4,683,202 and 4,682,195, each incorporatedherein by reference), or the synthesis of an oligonucleotide asdescribed in U.S. Pat. No. 5,645,897, incorporated herein by reference.A non-limiting example of a biologically produced nucleic acid includesa recombinant nucleic acid produced (i.e., replicated) in a living cell,such as a recombinant DNA vector replicated in bacteria (see forexample, Sambrook et al., 2001, incorporated herein by reference).

Oligonucleotide synthesis is well known to those of skill in the art.Various different mechanisms of oligonucleotide synthesis have beendisclosed in for example, U.S. Pat. Nos. 4,659,774, 4,816,571,5,141,813, 5,264,566, 4,959,463, 5,428,148, 5,554,744, 5,574,146,5,602,244, each of which is incorporated herein by reference.

Basically, chemical synthesis can be achieved by the diester method, thetriester method, polynucleotide phosphorylase method, and by solid-phasechemistry. The diester method was the first to be developed to a usablestate, primarily by Khorana and co-workers. (Khorana, 1979). The basicstep is the joining of two suitably protected deoxynucleotides to form adideoxynucleotide containing a phosphodiester bond.

The main difference between the diester and triester methods is thepresence in the latter of an extra protecting group on the phosphateatoms of the reactants and products (Itakura et al., 1975).Purifications are typically done in chloroform solutions. Otherimprovements in the method include (i) the block coupling of trimers andlarger oligomers, (ii) the extensive use of high-performance liquidchromatography for the purification of both intermediate and finalproducts, and (iii) solid-phase synthesis.

Polynucleotide phosphorylase method is an enzymatic method of DNAsynthesis that can be used to synthesize many useful oligonucleotides(Gillam et al., 1978; Gillam et al., 1979). Under controlled conditions,polynucleotide phosphorylase adds predominantly a single nucleotide to ashort oligonucleotide. Chromatographic purification allows the desiredsingle adduct to be obtained. At least a trimer is required to start theprocedure, and this primer must be obtained by some other method. Thepolynucleotide phosphorylase method works and has the advantage that theprocedures involved are familiar to most biochemists.

Solid-phase methods draw on technology developed for the solid-phasesynthesis of polypeptides. It has been possible to attach the initialnucleotide to solid support material and proceed with the stepwiseaddition of nucleotides. All mixing and washing steps are simplified,and the procedure becomes amenable to automation. These syntheses arenow routinely carried out using automatic nucleic acid synthesizers.

Phosphoramidite chemistry (Beaucage and Lyer, 1992) has become the mostwidely used coupling chemistry for the synthesis of oligonucleotides.Phosphoramidite synthesis of oligonucleotides involves activation ofnucleoside phosphoramidite monomer precursors by reaction with anactivating agent to form activated intermediates, followed by sequentialaddition of the activated intermediates to the growing oligonucleotidechain (generally anchored at one end to a suitable solid support) toform the oligonucleotide product.

Recombinant methods for producing nucleic acids in a cell are well knownto those of skill in the art. These include the use of vectors (viraland non-viral), plasmids, cosmids, and other vehicles for delivering anucleic acid to a cell, which may be the target cell (e.g., a cancercell) or simply a host cell (to produce large quantities of the desiredRNA molecule). Alternatively, such vehicles can be used in the contextof a cell free system so long as the reagents for generating the RNAmolecule are present. Such methods include those described in Sambrook,2003, Sambrook, 2001 and Sambrook, 1989, which are hereby incorporatedby reference.

In certain embodiments, nucleic acid molecules are not synthetic. Insome embodiments, the nucleic acid molecule has a chemical structure ofa naturally occurring nucleic acid and a sequence of a naturallyoccurring nucleic acid. In addition to the use of recombinanttechnology, such non-synthetic nucleic acids may be generatedchemically, such as by employing technology used for creatingoligonucleotides.

C. Isolation of Nucleic Acids

Nucleic acids may be isolated using techniques well known to those ofskill in the art, though in particular embodiments, methods forisolating small nucleic acid molecules, and/or isolating mRNA moleculescan be employed. Chromatography is a process often used to separate orisolate nucleic acids from protein or from other nucleic acids. Suchmethods can involve electrophoresis with a gel matrix, filter columns,alcohol precipitation, and/or other chromatography. If mRNA from cellsis to be used or evaluated, methods generally involve lysing the cellswith a chaotropic (e.g., guanidinium isothiocyanate) and/or detergent(e.g., N-lauroyl sarcosine) prior to implementing processes forisolating particular populations of RNA.

In particular methods for separating mRNA from other nucleic acids, agel matrix is prepared using polyacrylamide, though agarose can also beused. The gels may be graded by concentration or they may be uniform.Plates or tubing can be used to hold the gel matrix for electrophoresis.Usually one-dimensional electrophoresis is employed for the separationof nucleic acids. Plates are used to prepare a slab gel, while thetubing (glass or rubber, typically) can be used to prepare a tube gel.The phrase “tube electrophoresis” refers to the use of a tube or tubing,instead of plates, to form the gel. Materials for implementing tubeelectrophoresis can be readily prepared by a person of skill in the artor purchased.

Methods may involve the use of organic solvents and/or alcohol toisolate nucleic acids, particularly RNA used in methods and compositionsdisclosed herein. Some embodiments are described in U.S. patentapplication Ser. No. 10/667,126, which is hereby incorporated byreference. Generally, this disclosure provides methods for efficientlyisolating small RNA molecules from cells comprising: adding an alcoholsolution to a cell lysate and applying the alcohol/lysate mixture to asolid support before eluting the RNA molecules from the solid support.In some embodiments, the amount of alcohol added to a cell lysateachieves an alcohol concentration of about 55% to 60%. While differentalcohols can be employed, ethanol works well. A solid support may be anystructure, and it includes beads, filters, and columns, which mayinclude a mineral or polymer support with electronegative groups. Aglass fiber filter or column may work particularly well for suchisolation procedures.

In specific embodiments, RNA isolation processes include: a) lysingcells in the sample with a lysing solution comprising guanidinium,wherein a lysate with a concentration of at least about 1 M guanidiniumis produced; b) extracting RNA molecules from the lysate with anextraction solution comprising phenol; c) adding to the lysate analcohol solution for forming a lysate/alcohol mixture, wherein theconcentration of alcohol in the mixture is between about 35% to about70%; d) applying the lysate/alcohol mixture to a solid support; e)eluting the RNA molecules from the solid support with an ionic solution;and, f) capturing the RNA molecules. Typically the sample is dried downand resuspended in a liquid and volume appropriate for subsequentmanipulation.

D. Labels and Labeling Techniques

In some embodiments, nucleic acids and/or nucleic acid probes arelabeled. It is contemplated that nucleic acids may first be isolatedand/or purified prior to labeling. This may achieve a reaction that moreefficiently labels the nucleic acids, as opposed to other nucleic acidsin a sample in which the nucleic acids is not isolated or purified priorto labeling. In particular embodiments, the label is non-radioactive.Generally, nucleic acids may be labeled by adding labeled nucleotides(one-step process) or adding nucleotides and labeling the addednucleotides (two-step process).

1. Labeling Techniques

In some embodiments, nucleic acids are labeled by catalytically addingto the nucleic acid an already labeled nucleotide or nucleotides. One ormore labeled nucleotides can be added to nucleic acids molecules. SeeU.S. Pat. No. 6,723,509, which is hereby incorporated by reference.

In other embodiments, an unlabeled nucleotide(s) is catalytically addedto nucleic acids, and the unlabeled nucleotide is modified with achemical moiety that enables it to be subsequently labeled. In someembodiments, the chemical moiety is a reactive amine such that thenucleotide is an amine-modified nucleotide. Examples of amine-modifiednucleotides are well known to those of skill in the art, many beingcommercially available.

In contrast to labeling of cDNA during its synthesis, the issue forlabeling already synthesized nucleic acids is how to label the alreadyexisting molecule. Some aspects concern the use of an enzyme capable ofusing a di- or tri-phosphate ribonucleotide or deoxyribonucleotide as asubstrate for its addition to nucleic acids. Moreover, in specificembodiments, a modified di- or tri-phosphate ribonucleotide is added tothe 3′ end of a nucleic acid. The source of the enzyme is not limiting.Examples of sources for the enzymes include yeast, gram-negativebacteria such as E. coli, lactococcus lactis, and sheep pox virus.

Enzymes capable of adding such nucleotides include, but are not limitedto, poly(A) polymerase, terminal transferase, and polynucleotidephosphorylase. In specific embodiments, a ligase is contemplated as notbeing the enzyme used to add the label, and instead, a non-ligase enzymeis employed.

Terminal transferase may catalyze the addition of nucleotides to the 3′terminus of a nucleic acid. Polynucleotide phosphorylase can polymerizenucleotide diphosphates without the need for a primer.

2. Labels

Labels on nucleic acids or nucleic acid probes may be colorimetric(includes visible and UV spectrum, including fluorescent), luminescent,enzymatic, or positron emitting (including radioactive). The label maybe detected directly or indirectly. Radioactive labels include I¹²⁵,P³², P³³, and S³⁵. Examples of enzymatic labels include alkalinephosphatase, luciferase, horseradish peroxidase, and β-galactosidase.Labels can also be proteins with luminescent properties, e.g., greenfluorescent protein and phicoerythrin.

The colorimetric and fluorescent labels contemplated for use asconjugates include, but are not limited to, Alexa Fluor dyes, BODIPYdyes, such as BODIPY FL; Cascade Blue; Cascade Yellow; coumarin and itsderivatives, such as 7-amino-4-methylcoumarin, aminocoumarin andhydroxycoumarin; cyanine dyes, such as Cy3 and Cy5; eosins anderythrosins; fluorescein and its derivatives, such as fluoresceinisothiocyanate; macrocyclic chelates of lanthanide ions, such as QuantumDye™; Marina Blue; Oregon Green; rhodamine dyes, such as rhodamine red,tetramethylrhodamine and rhodamine 6G; Texas Red; fluorescent energytransfer dyes, such as thiazole orange-ethidium heterodimer; and, TOTAB.

Specific examples of dyes include, but are not limited to, thoseidentified above and the following: Alexa Fluor 350, Alexa Fluor 405,Alexa Fluor 430, Alexa Fluor 488, Alexa Fluor 500. Alexa Fluor 514,Alexa Fluor 532, Alexa Fluor 546, Alexa Fluor 555, Alexa Fluor 568,Alexa Fluor 594, Alexa Fluor 610, Alexa Fluor 633, Alexa Fluor 647,Alexa Fluor 660, Alexa Fluor 680, Alexa Fluor 700, and, Alexa Fluor 750;amine-reactive BODIPY dyes, such as BODIPY 493/503, BODIPY 530/550,BODIPY 558/568, BODIPY 564/570, BODIPY 576/589, BODIPY 581/591, BODIPY630/650, BODIPY 650/655, BODIPY FL, BODIPY R6G, BODIPY TMR, and,BODIPY-TR; Cy3, Cy5, 6-FAM, Fluorescein Isothiocyanate, HEX, 6-JOE,Oregon Green 488, Oregon Green 500, Oregon Green 514, Pacific Blue, REG,Rhodamine Green, Rhodamine Red, Renographin, ROX, SYPRO, TAMRA,2′,4′,5′,7′-Tetrabromosulfonefluorescein, and TET.

Specific examples of fluorescently labeled ribonucleotides include AlexaFluor 488-5-UTP, Fluorescein-12-UTP, BODIPY FL-14-UTP, BODIPYTMR-14-UTP, Tetramethylrhodamine-6-UTP, Alexa Fluor 546-14-UTP, TexasRed-5-UTP, and BODIPY TR-14-UTP. Other fluorescent ribonucleotidesinclude Cy3-UTP and Cy5-UTP.

Examples of fluorescently labeled deoxyribonucleotides includeDinitrophenyl (DNP)-11-dUTP, Cascade Blue-7-dUTP, Alexa Fluor488-5-dUTP, Fluorescein-12-dUTP, Oregon Green 488-5-dUTP, BODIPYFL-14-dUTP, Rhodamine Green-5-dUTP, Alexa Fluor 532-5-dUTP, BODIPYTMR-14-dUTP, Tetramethylrhodamine-6-dUTP, Alexa Fluor 546-14-dUTP, AlexaFluor 568-5-dUTP, Texas Red-12-dUTP, Texas Red-5-dUTP, BODIPYTR-14-dUTP, Alexa Fluor 594-5-dUTP, BODIPY 630/650-14-dUTP, BODIPY650/665-14-dUTP; Alexa Fluor 488-7-OBEA-dCTP, Alexa Fluor546-16-OBEA-dCTP, Alexa Fluor 594-7-OBEA-dCTP, and Alexa Fluor647-12-OBEA-dCTP.

It is contemplated that nucleic acids may be labeled with two differentlabels. Furthermore, fluorescence resonance energy transfer (FRET) maybe employed in disclosed methods (e.g., Klostermeier et al., 2002;Emptage, 2001; Didenko, 2001, each incorporated by reference).

Alternatively, the label may not be detectable per se, but indirectlydetectable or allowing for the isolation or separation of the targetednucleic acid. For example, the label could be biotin, digoxigenin,polyvalent cations, chelator groups and other ligands, include ligandsfor an antibody.

E. Visualization Techniques

A number of techniques for visualizing or detecting labeled nucleicacids are readily available. Such techniques include, microscopy,arrays, fluorometry, light cyclers or other real time PCR machines, FACSanalysis, scintillation counters, phosphoimagers, Geiger counters, MRI,CAT, antibody-based detection methods (Westerns, immunofluorescence,immunohistochemistry), histochemical techniques, HPLC (Griffey et al.,1997), spectroscopy, capillary gel electrophoresis (Cummins et al.,1996), spectroscopy; mass spectroscopy; radiological techniques; andmass balance techniques.

When two or more differentially colored labels are employed, fluorescentresonance energy transfer (FRET) techniques may be employed tocharacterize association of one or more nucleic acids. Furthermore, aperson of ordinary skill in the art is well aware of ways ofvisualizing, identifying, and characterizing labeled nucleic acids, andaccordingly, such protocols may be used. Examples of tools that may beused also include fluorescent microscopy, a BioAnalyzer, a plate reader,Storm (Molecular Dynamics), Array Scanner, FACS (fluorescent activatedcell sorter), or any instrument that has the ability to excite anddetect a fluorescent molecule.

F. Array Preparation and Screening

1. Array Preparation

Some embodiments involve the preparation and use of nucleic acid arraysor nucleic acid probe arrays, which are ordered macroarrays ormicroarrays of nucleic acid molecules (probes) that are fully or nearlycomplementary or identical to a plurality of nucleic acid molecules orprecursor nucleic acid molecules and that are positioned on a support orsupport material in a spatially separated organization. Macroarrays aretypically sheets of nitrocellulose or nylon upon which probes have beenspotted. Microarrays position the nucleic acid probes more densely suchthat up to 10,000 nucleic acid molecules can be fit into a regiontypically 1 to 4 square centimeters. Microarrays can be fabricated byspotting nucleic acid molecules, e.g., genes, oligonucleotides, etc.,onto substrates or fabricating oligonucleotide sequences in situ on asubstrate. Spotted or fabricated nucleic acid molecules can be appliedin a high density matrix pattern of up to about 30 non-identical nucleicacid molecules per square centimeter or higher, e.g. up to about 100 oreven 1000 per square centimeter. Microarrays typically use coated glassas the solid support, in contrast to the nitrocellulose-based materialof filter arrays. By having an ordered array of gene-complementingnucleic acid samples, the position of each sample can be tracked andlinked to the original sample. A variety of different array devices inwhich a plurality of distinct nucleic acid probes are stably associatedwith the surface of a solid support are known to those of skill in theart. Useful substrates for arrays include nylon, glass, metal, plastic,and silicon. Such arrays may vary in a number of different ways,including average probe length, sequence or types of probes, nature ofbond between the probe and the array surface, e.g. covalent ornon-covalent, and the like. The labeling and screening methods are notlimited by with respect to any parameter except that the probes detectnucleic acids; consequently, methods and compositions may be used with avariety of different types of nucleic acid arrays.

Representative methods and apparatuses for preparing a microarray havebeen described, for example, in U.S. Pat. Nos. 5,143,854; 5,202,231;5,242,974; 5,288,644; 5,324,633; 5,384,261; 5,405,783; 5,412,087;5,424,186; 5,429,807; 5,432,049; 5,436,327; 5,445,934; 5,468,613;5,470,710; 5,472,672; 5,492,806; 5,525,464; 5,503,980; 5,510,270;5,525,464; 5,527,681; 5,529,756; 5,532,128; 5,545,531; 5,547,839;5,554,501; 5,556,752; 5,561,071; 5,571,639; 5,580,726; 5,580,732;5,593,839; 5,599,695; 5,599,672; 5,610,287; 5,624,711; 5,631,134;5,639,603; 5,654,413; 5,658,734; 5,661,028; 5,665,547; 5,667,972;5,695,940; 5,700,637; 5,744,305; 5,800,992; 5,807,522; 5,830,645;5,837,196; 5,871,928; 5,847,219; 5,876,932; 5,919,626; 6,004,755;6,087,102; 6,368,799; 6,383,749; 6,617,112; 6,638,717; 6,720,138, aswell as WO 93/17126; WO 95/11995; WO 95/21265; WO 95/21944; WO 95/35505;WO 96/31622; WO 97/10365; WO 97/27317; WO 99/35505; WO 09923256; WO09936760; WO0138580; WO 0168255; WO 03020898; WO 03040410; WO 03053586;WO 03087297; WO 03091426; WO03100012; WO 04020085; WO 04027093; EP 373203; EP 785 280; EP 799 897 and UK 8 803 000, which are each hereinincorporated by reference.

It is contemplated that the arrays can be high density arrays, such thatthey contain 2, 20, 25, 50, 80, 100, or more, or any integer derivabletherein, different probes. It is contemplated that they may contain1000, 16,000, 65,000, 250,000 or 1,000,000 or more, or any interger orrange derivable therein, different probes. The probes can be directed totargets in one or more different organisms or cell types. In someembodiments, the oligonucleotide probes may range from 5 to 50, 5 to 45,10 to 40, 9 to 34, or 15 to 40 nucleotides in length. In certainembodiments, the oligonucleotide probes are 5, 10, 15, 20, 25, 30, 35,40 nucleotides in length, including all integers and ranges therebetween.

Moreover, the large number of different probes can occupy a relativelysmall area providing a high density array having a probe density ofgenerally greater than about 60, 100, 600, 1000, 5,000, 10,000, 40,000,100,000, or 400,000 different oligonucleotide probes per cm². Thesurface area of the array can be about or less than about 1, 1.6, 2, 3,4, 5, 6, 7, 8, 9, or 10 cm².

Moreover, a person of ordinary skill in the art could readily analyzedata generated using an array. Such protocols are disclosed herein ormay be found in, for example, WO 9743450; WO 03023058; WO 03022421; WO03029485; WO 03067217; WO 03066906; WO 03076928; WO 03093810; WO03100448A1, all of which are specifically incorporated by reference.

2. Sample Preparation

It is contemplated that the nucleic acids of a wide variety of samplescan be analyzed using arrays, nucleic acid probes, or array technology.While endogenous nucleic acids are contemplated for use withcompositions and methods disclosed herein, recombinant nucleicacids—including nucleic acids that are complementary or identical toendogenous nucleic acids—can also be handled and analyzed as describedherein. Samples may be biological samples, in which case, they can befrom biopsy, fine needle aspirates, exfoliates, blood, tissue, organs,semen, saliva, tears, other bodily fluid, hair follicles, skin, or anysample containing or constituting biological cells. In certainembodiments, samples may be, but are not limited to, fresh, frozen,fixed, formalin fixed, paraffin embedded, or formalin fixed and paraffinembedded. Alternatively, the sample may not be a biological sample, buta chemical mixture, such as a cell-free reaction mixture (which maycontain one or more biological enzymes).

After an array or a set of nucleic acid probes is prepared and thenucleic acids in the sample are labeled, the population of targetnucleic acids is contacted with the array or probes under hybridizationconditions, where such conditions can be adjusted, as desired, toprovide for an optimum level of specificity in view of the particularassay being performed. Suitable hybridization conditions are well knownto those of skill in the art and reviewed in Sambrook et al. (2001) andWO 95/21944. Of particular interest in embodiments is the use ofstringent conditions during hybridization. Stringent conditions areknown to those of skill in the art.

It is specifically contemplated that a single array or set of probes maybe contacted with multiple samples. The samples may be labeled withdifferent labels to distinguish the samples. For example, a single arraycan be contacted with a tumor tissue sample labeled with Cy3, and normaltissue sample labeled with Cy5. Differences between the samples forparticular nucleic acids corresponding to probes on the array can bereadily ascertained and quantified.

The small surface area of the array permits uniform hybridizationconditions, such as temperature regulation and salt content. Moreover,because of the small area occupied by the high density arrays,hybridization may be carried out in extremely small fluid volumes (e.g.,about 250 μl or less, including volumes of about or less than about 5,10, 25, 50, 60, 70, 80, 90, 100 μl, or any range derivable therein). Insmall volumes, hybridization may proceed very rapidly.

3. Differential Expression Analyses

Arrays can be used to detect differences between two samples.Specifically contemplated applications include identifying and/orquantifying differences between nucleic acids or mRNA from a sample thatis normal and from a sample that is not normal, between T-cell anergicand non T-cell anergic samples and/or a cancerous condition and anon-cancerous condition, or between two differently treated samples.Also, nucleic acids or mRNA may be compared between a sample believed tobe susceptible to a particular disease or condition and one believed tobe not susceptible or resistant to that disease or condition. A samplethat is not normal is one exhibiting phenotypic trait(s) of a disease orcondition or one believed to be not normal with respect to that diseaseor condition. It may be compared to a cell that is normal with respectto that disease or condition. Phenotypic traits include symptoms of, orsusceptibility to, a disease or condition of which a component is or mayor may not be genetic or caused by a hyperproliferative or neoplasticcell or cells.

An array comprises a solid support with nucleic acid probes attached tothe support. Arrays typically comprise a plurality of different nucleicacid probes that are coupled to a surface of a substrate in different,known locations. These arrays, also described as “microarrays” orcolloquially “chips” have been generally described in the art, forexample, U.S. Pat. Nos. 5,143,854, 5,445,934, 5,744,305, 5,677,195,6,040,193, 5,424,186 and Fodor et al., 1991), each of which isincorporated by reference in its entirety for all purposes. These arraysmay generally be produced using mechanical synthesis methods or lightdirected synthesis methods that incorporate a combination ofphotolithographic methods and solid phase synthesis methods. Techniquesfor the synthesis of these arrays using mechanical synthesis methods aredescribed in, e.g., U.S. Pat. No. 5,384,261, incorporated herein byreference in its entirety. Although a planar array surface is used incertain aspects, the array may be fabricated on a surface of virtuallyany shape or even a multiplicity of surfaces. Arrays may be nucleicacids on beads, gels, polymeric surfaces, fibers such as fiber optics,glass or any other appropriate substrate (see U.S. Pat. Nos. 5,770,358,5,789,162, 5,708,153, 6,040,193 and 5,800,992, each of which is herebyincorporated in its entirety). Arrays may be packaged in such a manneras to allow for diagnostics or other manipulation of an all inclusivedevice (see for example, U.S. Pat. Nos. 5,856,174 and 5,922,591, eachincorporated in its entirety by reference). See also U.S. patentapplication Ser. No. 09/545,207, filed Apr. 7, 2000, which isincorporated by reference in its entirety for additional informationconcerning arrays, their manufacture, and their characteristics,

Particularly, arrays can be used to evaluate samples with respect todiseases or conditions that include, but are not limited to: T-cellanergy, chronic pancreatitis; pancreatic cancer; AIDS, autoimmunediseases (rheumatoid arthritis, multiple sclerosis,diabetes—insulin-dependent and non-independent, systemic lupuserythematosus and Graves disease); cancer (e.g., malignant, benign,metastatic, precancer); cardiovascular diseases (heart disease orcoronary artery disease, stroke—ischemic and hemorrhagic, and rheumaticheart disease); diseases of the nervous system; and infection bypathogenic microorganisms (Athlete's Foot, Chickenpox, Common cold,Diarrheal diseases, Flu, Genital herpes, Malaria, Meningitis, Pneumonia,Sinusitis, Skin diseases, Strep throat, Tuberculosis, Urinary tractinfections, Vaginal infections, Viral hepatitis); inflammation (allergy,asthma); prion diseases (e.g., CJD, kuru, GSS, FFI).

Cancers that may be evaluated by the disclosed methods and compositionsinclude cells and cancer cells from the bladder, blood, bone, bonemarrow, brain, breast, colon, esophagus, gastrointestine, gum, head,kidney, liver, lung, nasopharynx, neck, ovary, prostate, skin, stomach,testis, tongue, or uterus. In addition, the cancer may specifically beof the following histological type, though it is not limited to these:neoplasm, malignant; carcinoma; carcinoma, undifferentiated; giant andspindle cell carcinoma; small cell carcinoma; papillary carcinoma;squamous cell carcinoma; lymphoepithelial carcinoma; basal cellcarcinoma; pilomatrix carcinoma; transitional cell carcinoma; papillarytransitional cell carcinoma; adenocarcinoma; gastrinoma, malignant;cholangiocarcinoma; hepatocellular carcinoma; combined hepatocellularcarcinoma and cholangiocarcinoma; trabecular adenocarcinoma; adenoidcystic carcinoma; adenocarcinoma in adenomatous polyp; adenocarcinoma,familial polyposis coli; solid carcinoma; carcinoid tumor, malignant;branchiolo-alveolar adenocarcinoma; papillary adenocarcinoma;chromophobe carcinoma; acidophil carcinoma; oxyphilic adenocarcinoma;basophil carcinoma; clear cell adenocarcinoma; granular cell carcinoma;follicular adenocarcinoma; papillary and follicular adenocarcinoma;nonencapsulating sclerosing carcinoma; adrenal cortical carcinoma;endometroid carcinoma; skin appendage carcinoma; apocrineadenocarcinoma; sebaceous adenocarcinoma; ceruminous adenocarcinoma;mucoepidermoid carcinoma; cystadenocarcinoma; papillarycystadenocarcinoma; papillary serous cystadenocarcinoma; mucinouscystadenocarcinoma; mucinous adenocarcinoma; signet ring cell carcinoma;infiltrating duct carcinoma; medullary carcinoma; lobular carcinoma;inflammatory carcinoma; paget's disease, mammary; acinar cell carcinoma;adenosquamous carcinoma; adenocarcinoma w/squamous metaplasia; thymoma,malignant; ovarian stromal tumor, malignant; thecoma, malignant;granulosa cell tumor, malignant; androblastoma, malignant; sertoli cellcarcinoma; leydig cell tumor, malignant; lipid cell tumor, malignant;paraganglioma, malignant; extra-mammary paraganglioma, malignant;pheochromocytoma; glomangiosarcoma; malignant melanoma; amelanoticmelanoma; superficial spreading melanoma; malig melanoma in giantpigmented nevus; epithelioid cell melanoma; blue nevus, malignant;sarcoma; fibrosarcoma; fibrous histiocytoma, malignant; myxosarcoma;liposarcoma; leiomyosarcoma; rhabdomyosarcoma; embryonalrhabdomyosarcoma; alveolar rhabdomyosarcoma; stromal sarcoma; mixedtumor, malignant; mullerian mixed tumor; nephroblastoma; hepatoblastoma;carcinosarcoma; mesenchymoma, malignant; brenner tumor, malignant;phyllodes tumor, malignant; synovial sarcoma; mesothelioma, malignant;dysgerminoma; embryonal carcinoma; teratoma, malignant; struma ovarii,malignant; choriocarcinoma; mesonephroma, malignant; hemangiosarcoma;hemangioendothelioma, malignant; kaposi's sarcoma; hemangiopericytoma,malignant; lymphangiosarcoma; osteosarcoma; juxtacortical osteosarcoma;chondrosarcoma; chondroblastoma, malignant; mesenchymal chondrosarcoma;giant cell tumor of bone; ewing's sarcoma; odontogenic tumor, malignant;ameloblastic odontosarcoma; ameloblastoma, malignant; ameloblasticfibrosarcoma; pinealoma, malignant; chordoma; glioma, malignant;ependymoma; astrocytoma; protoplasmic astrocytoma; fibrillaryastrocytoma; astroblastoma; glioblastoma; oligodendroglioma;oligodendroblastoma; primitive neuroectodermal; cerebellar sarcoma;ganglioneuroblastoma; neuroblastoma; retinoblastoma; olfactoryneurogenic tumor; meningioma, malignant; neurofibrosarcoma;neurilemmoma, malignant; granular cell tumor, malignant; malignantlymphoma; Hodgkin's disease; Hodgkin's lymphoma; paragranuloma;malignant lymphoma, small lymphocytic; malignant lymphoma, large cell,diffuse; malignant lymphoma, follicular; mycosis fungoides; otherspecified non-Hodgkin's lymphomas; malignant histiocytosis; multiplemyeloma; mast cell sarcoma; immunoproliferative small intestinaldisease; leukemia; lymphoid leukemia; plasma cell leukemia;erythroleukemia; lymphosarcoma cell leukemia; myeloid leukemia;basophilic leukemia; eosinophilic leukemia; monocytic leukemia; mastcell leukemia; megakaryoblastic leukemia; myeloid sarcoma; and hairycell leukemia. Moreover, T-cell anergy can be evaluated in precancers,such as metaplasia, dysplasia, and hyperplasia.

It is specifically contemplated that the disclosed methods andcompositions can be used to evaluate differences between stages ofdisease, such as between hyperplasia, neoplasia, pre-cancer and cancer,or between a primary tumor and a metastasized tumor. A tumor sample or acancer sample from a patient can include cancer cells or cells suspectedof being cancerous.

Moreover, it is contemplated that samples that have differences in theactivity of certain pathways may also be compared. These pathwaysinclude the following and those involving the following factors:antibody response, apoptosis, calcium/NFAT signaling, cell cycle, cellmigration, cell adhesion, cell division, cytokines and cytokinereceptors, drug metabolism, growth factors and growth factor receptors,inflammatory response, insulin signaling, NFκ-B signaling, angiogenesis,adipogenesis, cell adhesion, viral infection, bacterial infection,senescence, motility, glucose transport, stress response, oxidation,aging, telomere extension, telomere shortening, neural transmission,blood clotting, stem cell differentiation, G-Protein Coupled Receptor(GPCR) signaling, and p53 activation.

Cellular pathways that may be profiled also include but are not limitedto the following: any adhesion or motility pathway including but notlimited to those involving cyclic AMP, protein kinase A, G-proteincouple receptors, adenylyl cyclase, L-selectin, E-selectin, PECAM,VCAM-1, α-actinin, paxillin, cadherins, AKT, integrin-α, integrin-β,RAF-1, ERK, PI-3 kinase, vinculin, matrix metalloproteinases, RhoGTPases, p85, trefoil factors, profilin, FAK, MAP kinase, Ras, caveolin,calpain-1, calpain-2, epidermal growth factor receptor, ICAM-1, ICAM-2,cofilin, actin, gelsolin, RhoA, RAC1, myosin light chain kinase,platelet-derived growth factor receptor or ezrin; any apoptosis pathwayincluding but not limited to those involving AKT, Fas ligand, NFkappaB,caspase-9, PI3 kinase, caspase-3, caspase-7, ICAD, CAD, EndoG, GranzymeB, Bad, Bax, Bid, Bak, APAF-1, cytochrome C, p53, ATM, Bcl-2, PARP,Chkl, Chk2, p21, c-Jun, p73, RadSl, Mdm2, Rad50, c-Abl, BRCA-1,perforin, caspase-4, caspase-8, caspase-6, caspase-1, caspase-2,caspase-10, Rho, Jun kinase, Jun kinase kinase, Rip2, lamin-A, lamin-B1,lamin-B2, Fas receptor, H2O2, Granzyme A, NADPH oxidase, HMG2, CD4,CD28, CD3, TRADD, IKK, FADD, GADD45, DR3 death receptor, DR4/5 deathreceptor, FLIPs, APO-3, GRB2, SHC, ERK, MEK, RAF-1, cyclic AMP, proteinkinase A, E2F, retinoblastoma protein, Smac/Diablo, ACH receptor,14-3-3, FAK, SODD, TNF receptor, RIP, cyclin-Di, PCNA, Bcl-XL, PIP2,PIP3, PTEN, ATM, Cdc2, protein kinase C, calcineurin, IKKα, IKKβ, IKKγ,SOS-1, c-FOS, Traf-1, Traf-2, IκBβ or the proteasome; any cellactivation pathway including but not limited to those involving proteinkinase A, nitric oxide, caveolin-1, actin, calcium, protein kinase C,Cdc2, cyclin B, Cdc25, GRB2, SRC protein kinase, ADP-ribosylationfactors (ARFs), phospholipase D, AKAP95, p68, Aurora B, CDK1, Eg7,histone H3, PKAc, CD80, PI3 kinase, WASP, Arp2, Arp3, p16, p34, p20,PP2A, angiotensin, angiotensin-converting enzyme, protease-activatedreceptor-1, protease-activated receptor-4, Ras, RAF-1, PLCβ, PLCγ,COX-1, G-protein-coupled receptors, phospholipase A2, IP3, SUMO1, SUMO2/3, ubiquitin, Ran, Ran-GAP, Ran-GEF, p53, glucocorticoids,glucocorticoid receptor, components of the SWI/SNF complex, RanBP1,RanBP2, importins, exportins, RCC1, CD40, CD40 ligand, p38, IKKα, IKKβ,NFκB, TRAF2, TRAF3, TRAF5, TRAF6, IL-4, IL-4 receptor, CDK5, AP-1transcription factor, CD45, CD4, T cell receptors, MAP kinase, nervegrowth factor, nerve growth factor receptor, c-Jun, c-Fos, Jun kinase,GRB2, SOS-1, ERK-1, ERK, JAK2, STAT4, IL-12, IL-12 receptor, nitricoxide synthase, TYK2, IFNγ, elastase, IL-8, epithelins, IL-2, IL-2receptor, CD28, SMAD3, SMAD4, TGFβ or TGFβ receptor; any cell cycleregulation, signaling or differentiation pathway including but notlimited to those involving TNFs, SRC protein kinase, Cdc2, cyclin B,Grb2, Sos-1, SHC, p68, Aurora kinases, protein kinase A, protein kinaseC, Eg7, p53, cyclins, cyclin-dependent kinases, neural growth factor,epidermal growth factor, retinoblastoma protein, ATF-2, ATM, ATR, AKT,CHK1, CHK2, 14-3-3, WEE1, CDC25 CDC6, Origin Recognition Complexproteins, p15, p16, p2′7, p21, ABL, c-ABL, SMADs, ubiquitin, SUMO, heatshock proteins, Wnt, GSK-3, angiotensin, p73 any PPAR, TGFα, TGFβ, p300,MDM2, GADD45, Notch, cdc34, BRCA-1, BRCA-2, SKP1, the proteasome, CUL1,E2F, p107, steroid hormones, steroid hormone receptors, IκBα, IκBβ,Sin3A, heat shock proteins, Ras, Rho, ERKs, IKKs, PI3 kinase, Bcl-2,Bax, PCNA, MAP kinases, dynein, RhoA, PKAc, cyclin AMP, FAK, PIP2, PIP3,integrins, thrombopoietin, Fas, Fas ligand, PLK3, MEKs, JAKs, STATs,acetylcholine, paxillin calcineurin, p38, importins, exportins, Ran,Rad50, Rad51, DNA polymerase, RNA polymerase, Ran-GAP, Ran-GEF, NuMA,Tpx2, RCC1, Sonic Hedgehog, Crml, Patched (Ptc-1), MPF, CaM kinases,tubulin, actin, kinetochore-associated proteins, centromere-bindingproteins, telomerase, TERT, PP2A, c-MYC, insulin, T cell receptors, Bcell receptors, CBP, IKβ, NFκB, RAC1, RAF1, EPO, diacylglycerol, c-Jun,c-Fos, Jun kinase, hypoxia-inducible factors, GATA4, β-catenin,α-catenin, calcium, arrestin, survivin, caspases, procaspases, CREB,CREM, cadherins, PECAMs, corticosteroids, colony-stimulating factors,calpains, adenylyl cyclase, growth factors, nitric oxide, transmembranereceptors, retinoids, G-proteins, ion channels, transcriptionalactivators, transcriptional coactivators, transcriptional repressors,interleukins, vitamins, interferons, transcriptional corepressors, thenuclear pore, nitrogen, toxins, proteolysis, or phosphorylation; or anymetabolic pathway including but not limited to those involving thebiosynthesis of amino acids, oxidation of fatty acids, biosynthesis ofneurotransmitters and other cell signaling molecules, biosynthesis ofpolyamines, biosynthesis of lipids and sphingolipids, catabolism ofamino acids and nutrients, nucleotide synthesis, eicosanoids, electrontransport reactions, ER-associated degradation, glycolysis,fibrinolysis, formation of ketone bodies, formation of phagosomes,cholesterol metabolism, regulation of food intake, energy homeostasis,prothrombin activation, synthesis of lactose and other sugars,multi-drug resistance, biosynthesis of phosphatidylcholine, theproteasome, amyloid precursor protein, Rab GTPases, starch synthesis,glycosylation, synthesis of phoshoglycerides, vitamins, the citric acidcycle, IGF-1 receptor, the urea cycle, vesicular transport, or salvagepathways. It is further contemplated that the disclosed nucleic acidsmolecules can be employed in diagnostic and therapeutic methods withrespect to any of the above pathways or factors. Thus, in someembodiments, a T-cell anergy gene may be differentially expressed withrespect to one or more of the above pathways or factors.

Phenotypic traits also include characteristics such as longevity,morbidity, appearance (e.g., baldness, obesity), strength, speed,endurance, fertility, susceptibility or receptivity to particular drugsor therapeutic treatments (drug efficacy), and risk of drug toxicity.Samples that differ in these phenotypic traits may also be evaluatedusing the arrays and methods described.

In certain embodiments, nucleic acid or mRNA profiles may be generatedto evaluate and correlate those profiles with pharmacokinetics. Forexample, nucleic acid or mRNA profiles may be created and evaluated forpatient tumor and blood samples prior to the patient being treated orduring treatment to determine if there are nucleic acid or mRNAs whoseexpression correlates with the outcome of the patient. Identification ofdifferential nucleic acid or mRNAs can lead to a diagnostic assayinvolving them that can be used to evaluate tumor and/or blood samplesto determine what drug regimen the patient should be provided. Inaddition, identification of differential nucleic acid or mRNAs can beused to identify or select patients suitable for a particular clinicaltrial. If a nucleic acid or mRNA profile is determined to be correlatedwith drug efficacy or drug toxicity, such may be relevant to whetherthat patient is an appropriate patient for receiving a drug or for aparticular dosage of a drug.

G. Nucleic Acid Amplification

Quantitative and semiquantive PCR. These amplification methods may beemployed to evaluate, assay or measure T-cell anergy gene expressionlevel. Semiquantitave PCR is used as described by (Murphy et al., 1993;Salvi et al., 1995). These amplification methods may involve primers orprimer pairs that are complementary to the sequence to be detected.

H. Other Nucleic Acid Assays

In addition to the use of arrays and microarrays, it is contemplatedthat a number of different assays could be employed to analyze genes,expressed genes, genes that regulate or are regulated by the same, theiractivities, and their effects. Such assays include, but are not limitedto, nucleic acid amplification, polymerase chain reaction, quantitativePCR, RT-PCR, in situ hybridization, Northern hybridization,hybridization protection assay (HPA), branched DNA (bDNA) assay, rollingcircle amplification (RCA), single molecule hybridization detection,Invader assay, and/or Bridge Litigation Assay.

I. Immunoassays

As an alternative to, or in tandem with nucleic acid detection methods,isolated cells from patient samples or biopsies can be lysed and T-cellanergy gene protein levels can be determined by Western blotting methodswith T-cell anergy gene specific antibodies. Antibodies of the presentinvention can be used in characterizing the T-cell anergy gene proteincontent of tissues or cells in bodily fluids such as blood,cerebrospinal fluid, urine, prostate fluid or semen through techniquessuch as RIAs, ELISAs and Western blotting.

Immunoassays can be classified according to the assay type, assay methodand endpoint labeling method. These three major criteria forclassification that have the greatest influence on the performance oftest are, i) the use of a limited (type II) or excessive reagent format(type I), ii) the use of a homogeneous and heterogeneous format, iii)the use of a label or unlabelled assay format and the choice of label.It is contemplated that all these kinds of immunoassays may be employed.

In Type I assay format, where antigen binds to an excess of antibody,the most common method is sandwich assay. In this approach, the firstantibody (capture Ab) in excess is coupled to a solid phase. The boundantigen is then detected with a second antibody (indicator Ab) labeledwith various indicators such as enzymes, fluorophores, radioisotopes,particles, etc. In this assay, the amount of indicator antibody capturedon the solid phase is directory proportional to the amount of antigen inthe sample.

In some embodiments, an ELISA assay is particularly contemplated. Forexample, antibodies to T-cell anergy genes may be immobilized onto aselected surface, for example, a surface such as a microtiter well, amembrane, a filter, a bead or a dipstick. After washing to removeincompletely adsorbed material, it is desirable to bind or coat thesurface with a non-specific agent that is known to be antigenicallyneutral with regard to the test sample, e.g., bovine serum albumin(BSA), casein or solutions of powdered milk. This allows for blocking ofnon-specific adsorption sites on the immobilizing surface and thusreduces the background caused by non-specific binding of antibody toantigen on the surface.

After binding of antibody to the surface and coating, the surface isexposed to lysed patient tumor sample, blood, blood plasma, blood serum,urine, prostate fluid or semen. Following formation of specificimmunocomplexes between antigens in the blood and the antibody, andsubsequent washing, the occurrence and even amount of immunocomplexformation may be determined by subjecting the same to a second antibodyhaving specificity for the antigen. Appropriate conditions preferablyinclude diluting the sample with diluents such as BSA, bovine gammaglobulin (BGG) and phosphate buffered saline (PBS)/Tween. These addedagents also tend to assist in the reduction of non-specific background.The detecting antibody is then allowed to incubate for from about 2 toabout 4 hr, at temperatures preferably on the order of about 25° toabout 27° C. Following incubation, the surface is washed so as to removenon-immunocomplexed material. A preferred washing procedure includeswashing with a solution such as PBS/Tween or borate buffer.

To provide a detecting means, the second antibody may have an associatedlabel, e.g., an enzyme that will generate a color development uponincubating with an appropriate chromogenic substrate. Thus, for example,one will desire to contact and incubate the second antibody for a periodof time and under conditions which favor the development ofimmunocomplex formation (e.g., incubation for 2 hr at room temperaturein a PBS-containing solution such as PBS/Tween®.).

After incubation with the second antibody, and subsequent to washing toremove unbound material, the amount of label may be quantified (e.g., byincubation with a chromogenic substrate such as urea and bromocresolpurple or 2,2′-azino-di-(3-ethyl-benzthiazoline)-6-sulfonic acid (ABTS)and hydrogen peroxide in the case of peroxidase as the enzyme label).Quantitation is then achieved by measuring the label, e.g., degree ofcolor generation, e.g., using a visible spectrum spectrophotometer.

Other potential labels include radiolabels, fluorescent labels, dyes andchemiluminescent molecules (e.g., luciferase).

In Type II assay formats, a limited amount of antibody is used(insufficient to bind the entire antigen) a prefixed amount of labeledantigen competes with the unlabeled antigen in test sample for a limitednumber of antibody binding sites. The concentration of unlabeled antigenin specimen can be determined from the portion of labeled antigen thatis bound to the antibody. Since most analyte molecules are not enoughlarge to provide two different epitopes in this method, the responsewill be inversely proportional to the concentration of antigen in theunknown.

The use of either competitive or immunometric assays requiresdifferentiation of bound from free label. This can be archived either byseparating bound from free label using a means of removing antibody(heterogeneous) or modulation of signal of the label when antigen isbound to antibody compared to when it is free (homogeneous).

Most solid phase immunoassays belong to the Heterogeneous Assaycategory. There are many ways of separating bound from free label suchas precipitation of antibody, chromatographic method, and solid phasecoupling antibody. Homogeneous assays do not require any of separationstep to distinguish antigen bound antibody from free antibody. It has anadvantage in automation, and typically is faster, easier to perform, andmore cost-effective, but its specificity and sensitivity are lower.

Contacting the chosen biological sample with the first antibody undereffective conditions and for a period of time sufficient to allow theformation of immune complexes (primary immune complexes) is generally amatter of simply adding the antibody composition to the sample andincubating the mixture for a period of time long enough for theantibodies to form immune complexes with, i.e., to bind to, anyanti-T-cell anergy protein complex. After this time, the sample-antibodycomposition, such as a tissue section, ELISA plate, dot blot or westernblot, will generally be washed to remove any non-specifically boundantibody species, allowing only those antibodies specifically boundwithin the primary immune complexes to be detected.

In general, the detection of immunocomplex formation is well known inthe art and may be achieved through the application of numerousapproaches. These methods are generally based upon the detection of alabel or marker, such as any of those radioactive, fluorescent,biological and enzymatic tags. U.S. patents concerning the use of suchlabels include U.S. Pat. Nos. 3,817,837; 3,850,752; 3,939,350;3,996,345; 4,277,437; 4,275,149 and 4,366,241, each incorporated hereinby reference. Of course, one may find additional advantages through theuse of a secondary binding ligand such as a second antibody and/or abiotin/avidin ligand binding arrangement, as is known in the art. Allprior assays to detect immunocomplexes are based on autologous complexesgenerated by the patient's own antibodies and antigen. The presentinvention is different in that the assays of the present inventiondetect immunocomplexes as a result of a therapeutic approach.

The antigen, antibody or antigen:antibody complex employed in thedetection may itself be linked to a detectable label, wherein one wouldthen simply detect this label, thereby allowing the amount of theprimary immune complexes in the composition to be determined.Alternatively, the first antibody that becomes bound within the primaryimmune complexes may be detected by means of a second binding ligandthat has binding affinity for the antibody. In these cases, the secondbinding ligand may be linked to a detectable label. The second bindingligand is itself often an antibody, which may thus be termed a“secondary” antibody. The primary immune complexes are contacted withthe labeled, secondary binding ligand, or antibody, under effectiveconditions and for a period of time sufficient to allow the formation ofsecondary immune complexes. The secondary immune complexes are thengenerally washed to remove any non-specifically bound labeled secondaryantibodies or ligands, and the remaining label in the secondary immunecomplexes is then detected.

Further methods include the detection of primary immune complexes by atwo step approach. A second binding ligand, such as an antibody, thathas binding affinity for the antibody is used to form secondary immunecomplexes, as described above. After washing, the secondary immunecomplexes are contacted with a third binding ligand or antibody that hasbinding affinity for the second antibody, again under effectiveconditions and for a period of time sufficient to allow the formation ofimmune complexes (tertiary immune complexes). The third ligand orantibody is linked to a detectable label, allowing detection of thetertiary immune complexes thus formed. This system may provide forsignal amplification if this is desired.

As detailed above, immunoassays, in their most simple and/or directsense, are binding assays. Certain preferred immunoassays are thevarious types of enzyme linked immunosorbent assays (ELISAs) and/orradioimmunoassays (RIA) known in the art. Immunohistochemical detectionusing tissue sections is also particularly useful. However, it will bereadily appreciated that detection is not limited to such techniques,and/or western blotting, dot blotting, FACS analyses, and/or the likemay also be used.

Immunoassays encompassed by the present invention include, but are notlimited to, those described in U.S. Pat. No. 4,367,110 (doublemonoclonal antibody sandwich assay) and U.S. Pat. No. 4,452,901 (westernblot). Other assays include immunoprecipitation of labeled ligands andimmunocytochemistry, both in vitro and in vivo.

Immunoassays generally are binding assays. Certain useful immunoassaysare the various types of enzyme linked immunosorbent assays (ELISAs) andradioimmunoassays (RIA) known in the art. Immunohistochemical detectionusing tissue sections is also particularly useful.

In one exemplary ELISA, the antibodies are immobilized on a selectedsurface, such as a well in a polystyrene microtiter plate, dipstick, orcolumn support. Then, a test composition suspected of containing thedesired antigen, such as a clinical sample, is added to the wells. Afterbinding and washing to remove non-specifically bound immune complexes,the bound antigen may be detected. Detection is generally achieved bythe addition of another antibody, specific for the desired antigen, thatis linked to a detectable label. This type of ELISA is known as a“sandwich ELISA”. Detection also may be achieved by the addition of asecond antibody specific for the desired antigen, followed by theaddition of a third antibody that has binding affinity for the secondantibody, with the third antibody being linked to a detectable label.

Variations on ELISA techniques are known to those of skill in the art.In one such variation, the samples suspected of containing the desiredantigen are immobilized onto the well surface and then contacted withthe antibodies of the invention. After binding and appropriate washing,the bound immune complexes are detected. Where the initial antigenspecific antibodies are linked to a detectable label, the immunecomplexes may be detected directly. Again, the immune complexes may bedetected using a second antibody that has binding affinity for the firstantigen specific antibody, with the second antibody being linked to adetectable label.

Competition ELISAs are also possible in which test samples compete forbinding with known amounts of labeled antigens or antibodies. The amountof reactive species in the unknown sample is determined by mixing thesample with the known labeled species before or during incubation withcoated wells. The presence of reactive species in the sample acts toreduce the amount of labeled species available for binding to the welland thus reduces the ultimate signal.

Irrespective of the format employed, ELISAs have certain features incommon, such as coating, incubating or binding, washing to removenon-specifically bound species, and detecting the bound immunecomplexes. These are described as below.

Antigen or antibodies may also be linked to a solid support, such as inthe form of plate, beads, dipstick, membrane, or column matrix, and thesample to be analyzed is applied to the immobilized antigen or antibody.In coating a plate with either antigen or antibody, one will generallyincubate the wells of the plate with a solution of the antigen orantibody, either overnight or for a specified period. The wells of theplate will then be washed to remove incompletely-adsorbed material. Anyremaining available surfaces of the wells are then “coated” with anonspecific protein that is antigenically neutral with regard to thetest antisera. These include bovine serum albumin (BSA), casein, andsolutions of milk powder. The coating allows for blocking of nonspecificadsorption sites on the immobilizing surface and thus reduces thebackground caused by nonspecific binding of antisera onto the surface.

In ELISAs, it is more customary to use a secondary or tertiary detectionmeans rather than a direct procedure. Thus, after binding of the antigenor antibody to the well, coating with a non-reactive material to reducebackground, and washing to remove unbound material, the immobilizingsurface is contacted with the clinical or biological sample to be testedunder conditions effective to allow immune complex (antigen/antibody)formation. Detection of the immune complex then requires a labeledsecondary binding ligand or antibody, or a secondary binding ligand orantibody in conjunction with a labeled tertiary antibody or thirdbinding ligand.

“Under conditions effective to allow immune complex (antigen/antibody)formation” means that the conditions typically include diluting theantigens and antibodies with solutions such as BSA, bovine gammaglobulin (BGG) and phosphate buffered saline (PBS)/TWEEN (polysorbate).These added agents also tend to assist in the reduction of nonspecificbackground.

The suitable conditions also mean that the incubation is at atemperature and for a period of time sufficient to allow effectivebinding. Incubation steps are typically from about 1 to 2 to 4 hours, attemperatures that may be on the order of 25° C. to 27° C., or may beovernight at about 4° C. or so.

After all incubation steps in an ELISA are followed, the contactedsurface is washed so as to remove non-complexed material. Washing oftenincludes washing with a solution of PBS/TWEEN,® or borate buffer.Following the formation of specific immune complexes between the testsample and the originally bound material, and subsequent washing, theoccurrence of even minute amounts of immune complexes may be determined.

To provide a detecting means, the second or third antibody will have anassociated label to allow detection. In some embodiments, this will bean enzyme that will generate color development upon incubating with anappropriate chromogenic substrate. Thus, for example, one will desire tocontact and incubate the first or second immune complex with a urease,glucose oxidase, alkaline phosphatase, or hydrogen peroxidase-conjugatedantibody for a period of time and under conditions that favor thedevelopment of further immune complex formation, e.g., incubation for 2hours at room temperature in a PBS-containing solution such asPBS-TWEEN™.

After incubation with the labeled antibody, and subsequent to washing toremove unbound material, the amount of label is quantified, e.g., byincubation with a chromogenic substrate such as urea and bromocresolpurple or 2,2′-azino-di-(3-ethyl-benzthiazoline-6-sulfonic acid [ABTS]and H₂O₂, in the case of peroxidase as the enzyme label. Quantificationis then achieved by measuring the degree of color generation, e.g.,using a visible spectra spectrophotometer.

Alternatively, the label may be a chemiluminescent one. The use of suchlabels is described in U.S. Pat. Nos. 5,310,687, 5,238,808 and 5,221,605

It also is contemplated that the above reagents maybe packaged in a kitthat may be produced commercially to measure the soluble antigens,antibodies or antibody:antigen complexes described herein.

J. Antibodies and Antibody-Like Molecules

In certain aspects, one or more antibodies or antibody-like molecules(e.g., polypeptides comprising antibody CDR domains) may be obtained orproduced which have a specificity for a T-cell anergy gene product ofTable 2 or a T-cell anergy related cell surface receptor selected fromSemaphorin 7A (Sema7A), Class-I-MHC-restricted T cell associatedmolecule (Crtam), lymphocyte-activation gene 3 (Lag3), tumor necrosisfactor receptor superfamily member 9 (Tnfrsf9, also known as 4-1BB),Neuritin (Nrn1), CLIP (CD74), Tnfsf11 (RANKL, CD254), prostaglandin F2receptor inhibitor (Ptgfrn, CD9-P1, CD315) or oxidized low densitylipoprotein receptor 1 (LOX1, OLR1). These antibodies may be used invarious diagnostic or therapeutic applications described herein.

As used herein, the term “antibody” is intended to refer broadly to anyimmunologic binding agent such as IgG, IgM, IgA, IgD and IgE as well aspolypeptides comprising antibody CDR domains that retain antigen bindingactivity. Thus, the term “antibody” is used to refer to anyantibody-like molecule that has an antigen binding region, and includesantibody fragments such as Fab′, Fab, F(ab′)₂, single domain antibodies(DABs), Fv, scFv (single chain Fv), and polypeptides with antibody CDRs,scaffolding domains that display the CDRs (e.g., anticalins) or ananobody. For example, the nanobody can be antigen-specific VHH (e.g., arecombinant VHH) from a camelid IgG2 or IgG3, or a CDR-displaying framefrom such camelid Ig. The techniques for preparing and using variousantibody-based constructs and fragments are well known in the art. Meansfor preparing and characterizing antibodies are also well known in theart (See, e.g., Antibodies: A Laboratory Manual, Cold Spring HarborLaboratory, 1988; incorporated herein by reference).

“Mini-antibodies” or “minibodies” are also contemplated for use withembodiments. Minibodies are sFv polypeptide chains which includeoligomerization domains at their C-termini, separated from the sFv by ahinge region. Pack et al. (1992). The oligomerization domain comprisesself-associating α-helices, e.g., leucine zippers, that can be furtherstabilized by additional disulfide bonds. The oligomerization domain isdesigned to be compatible with vectorial folding across a membrane, aprocess thought to facilitate in vivo folding of the polypeptide into afunctional binding protein. Generally, minibodies are produced usingrecombinant methods well known in the art. See, e.g., Pack et al.(1992); Cumber et al. (1992).

Antibody-like binding peptidomimetics are also contemplated inembodiments. Liu et al. (2003) describe “antibody like bindingpeptidomimetics” (ABiPs), which are peptides that act as pared-downantibodies and have certain advantages of longer serum half-life as wellas less cumbersome synthesis methods.

Alternative scaffolds for antigen binding peptides, such as CDRs arealso available and can be used to generate binding molecules for aT-cell anergy gene product of Table 2 or a T-cell anergy related cellsurface receptor selected from Semaphorin 7A (Sema7A),Class-I-MHC-restricted T cell associated molecule (Crtam),lymphocyte-activation gene 3 (Lag3), tumor necrosis factor receptorsuperfamily member 9 (Tnfrsf9, also known as 4-1BB), Neuritin (Nrn1),CLIP (CD74), Tnfsf11 (RANKL, CD254), prostaglandin F2 receptor inhibitor(Ptgfrn, CD9-P1, CD315) or oxidized low density lipoprotein receptor 1(LOX1, OLR1) in accordance with the embodiments. Generally, a personskilled in the art knows how to determine the type of protein scaffoldon which to graft at least one of the CDRs arising from the originalantibody. More particularly, it is known that to be selected suchscaffolds must meet the greatest number of criteria as follows (Skerra,2000): good phylogenetic conservation; known three-dimensional structure(as, for example, by crystallography, NMR spectroscopy or any othertechnique known to a person skilled in the art); small size; few or nopost-transcriptional modifications; and/or easy to produce, express andpurify.

The origin of such protein scaffolds can be, but is not limited to, thestructures selected among: fibronectin and preferentially fibronectintype III domain 10, lipocalin, anticalin (Skerra, 2001), protein Zarising from domain B of protein A of Staphylococcus aureus, thioredoxinA or proteins with a repeated motif such as the “ankyrin repeat” (Kohlet al., 2003), the “armadillo repeat”, the “leucine-rich repeat” and the“tetratricopeptide repeat”. For example, anticalins or lipocalinderivatives are a type of binding proteins that have affinities andspecificities for various target molecules and can be used as bindingmolecules for a T-cell anergy gene product of Table 2 or a T-cell anergyrelated cell surface receptor selected from Semaphorin 7A (Sema7A),Class-I-MHC-restricted T cell associated molecule (Crtam),lymphocyte-activation gene 3 (Lag3), tumor necrosis factor receptorsuperfamily member 9 (Tnfrsf9, also known as 4-1BB), Neuritin (Nrn1),CLIP (CD74), Tnfsf11 (RANKL, CD254), prostaglandin F2 receptor inhibitor(Ptgfrn, CD9-P1, CD315) or oxidized low density lipoprotein receptor 1(LOX1, OLR1). Such proteins are described in US Patent Publication Nos.20100285564, 20060058510, 20060088908, 20050106660, and PCT PublicationNo. WO2006/056464, incorporated herein by reference.

Scaffolds derived from toxins such as, for example, toxins fromscorpions, insects, plants, mollusks, etc., and the protein inhibitersof neuronal NO synthase (PIN) may also be used in certain aspects.

Monoclonal antibodies (MAbs) are recognized to have certain advantages,e.g., reproducibility and large-scale production. Embodiments includemonoclonal antibodies of the human, murine, monkey, rat, hamster, rabbitand chicken origin.

“Humanized” antibodies are also contemplated, as are chimeric antibodiesfrom mouse, rat, or other species, bearing human constant and/orvariable region domains, bispecific antibodies, recombinant andengineered antibodies and fragments thereof. As used herein, the term“humanized” immunoglobulin refers to an immunoglobulin comprising ahuman framework region and one or more CDR's from a non-human (usually amouse or rat) immunoglobulin. The non-human immunoglobulin providing theCDR's is called the “donor” and the human immunoglobulin providing theframework is called the “acceptor”. A “humanized antibody” is anantibody comprising a humanized light chain and a humanized heavy chainimmunoglobulin.

1. Methods for Generating Antibodies

Methods for generating antibodies (e.g., monoclonal antibodies and/ormonoclonal antibodies) are known in the art. Briefly, a polyclonalantibody is prepared by immunizing an animal with a T-cell anergy geneproduct of Table 2 or a T-cell anergy related cell surface receptorselected from Semaphorin 7A (Sema7A), Class-I-MHC-restricted T cellassociated molecule (Crtam), lymphocyte-activation gene 3 (Lag3), tumornecrosis factor receptor superfamily member 9 (Tnfrsf9, also known as4-1BB), Neuritin (Nrn1), CLIP (CD74), Tnfsf11 (RANKL, CD254),prostaglandin F2 receptor inhibitor (Ptgfrn, CD9-P1, CD315) or oxidizedlow density lipoprotein receptor 1 (LOX1, OLR1) polypeptide or a portionthereof in accordance with embodiments and collecting antisera from thatimmunized animal.

A wide range of animal species can be used for the production ofantisera. Typically the animal used for production of antisera is arabbit, a mouse, a rat, a hamster, a guinea pig or a goat. The choice ofanimal may be decided upon the ease of manipulation, costs or thedesired amount of sera, as would be known to one of skill in the art. Itwill be appreciated that antibodies can also be produced transgenicallythrough the generation of a mammal or plant that is transgenic for theimmunoglobulin heavy and light chain sequences of interest andproduction of the antibody in a recoverable form therefrom. Inconnection with the transgenic production in mammals, antibodies can beproduced in, and recovered from, the milk of goats, cows, or othermammals. See, e.g., U.S. Pat. Nos. 5,827,690, 5,756,687, 5,750,172, and5,741,957.

As is also well known in the art, the immunogenicity of a particularimmunogen composition can be enhanced by the use of non-specificstimulators of the immune response, known as adjuvants. Suitableadjuvants include any acceptable immunostimulatory compound, such ascytokines, chemokines, cofactors, toxins, plasmodia, syntheticcompositions or vectors encoding such adjuvants.

Adjuvants that may be used in accordance with embodiments include, butare not limited to, IL-1, IL-2, IL-4, IL-7, IL-12, -interferon, GMCSP,BCG, aluminum hydroxide, MDP compounds, such as thur-MDP and nor-MDP,CGP (MTP-PE), lipid A, and monophosphoryl lipid A (MPL). RIBI, whichcontains three components extracted from bacteria, MPL, trehalosedimycolate (TDM) and cell wall skeleton (CWS) in a 2% squalene/Tween 80emulsion is also contemplated. MHC antigens may even be used. Exemplaryadjuvants may include complete Freund's adjuvant (a non-specificstimulator of the immune response containing killed Mycobacteriumtuberculosis), incomplete Freund's adjuvants and/or aluminum hydroxideadjuvant.

In addition to adjuvants, it may be desirable to coadminister biologicresponse modifiers (BRM), which have been shown to upregulate T cellimmunity or downregulate suppressor cell activity. Such BRMs include,but are not limited to, Cimetidine (CIM; 1200 mg/d) (Smith/Kline, PA);low-dose Cyclophosphamide (CYP; 300 mg/m2) (Johnson/Mead, NJ), cytokinessuch as -interferon, IL-2, or IL-12 or genes encoding proteins involvedin immune helper functions, such as B-7.

The amount of immunogen composition used in the production of antibodiesvaries upon the nature of the immunogen as well as the animal used forimmunization. A variety of routes can be used to administer theimmunogen including but not limited to subcutaneous, intramuscular,intradermal, intraepidermal, intravenous and intraperitoneal. Theproduction of antibodies may be monitored by sampling blood of theimmunized animal at various points following immunization.

A second, booster dose (e.g., provided in an injection), may also begiven. The process of boosting and titering is repeated until a suitabletiter is achieved. When a desired level of immunogenicity is obtained,the immunized animal can be bled and the serum isolated and stored,and/or the animal can be used to generate MAbs.

For production of rabbit polyclonal antibodies, the animal can be bledthrough an ear vein or alternatively by cardiac puncture. The removedblood is allowed to coagulate and then centrifuged to separate serumcomponents from whole cells and blood clots. The serum may be used as isfor various applications or else the desired antibody fraction may bepurified by well-known methods, such as affinity chromatography usinganother antibody, a peptide bound to a solid matrix, or by using, e.g.,protein A or protein G chromatography, among others.

MAbs may be readily prepared through use of well-known techniques, suchas those exemplified in U.S. Pat. No. 4,196,265, incorporated herein byreference. Typically, this technique involves immunizing a suitableanimal with a selected immunogen composition, e.g., a purified orpartially purified protein, polypeptide, peptide or domain, be it awild-type or mutant composition. The immunizing composition isadministered in a manner effective to stimulate antibody producingcells.

The methods for generating monoclonal antibodies (MAbs) generally beginalong the same lines as those for preparing polyclonal antibodies. Insome embodiments, Rodents such as mice and rats are used in generatingmonoclonal antibodies. In some embodiments, rabbit, sheep or frog cellsare used in generating monoclonal antibodies. The use of rats is wellknown and may provide certain advantages (Goding, 1986, pp. 60 61). Mice(e.g., BALB/c mice) are routinely used and generally give a highpercentage of stable fusions.

The animals are injected with antigen, generally as described above. Theantigen may be mixed with adjuvant, such as Freund's complete orincomplete adjuvant. Booster administrations with the same antigen orDNA encoding the antigen may occur at approximately two-week intervals.

Following immunization, somatic cells with the potential for producingantibodies, specifically B lymphocytes (B cells), are selected for usein the MAb generating protocol. These cells may be obtained frombiopsied spleens, tonsils or lymph nodes, or from a peripheral bloodsample. Generally, spleen cells are a rich source of antibody-producingcells that are in the dividing plasmablast stage. Typically, peripheralblood cells may be readily obtained, as peripheral blood is easilyaccessible.

In some embodiments, a panel of animals will have been immunized and thespleen of an animal with the highest antibody titer will be removed andthe spleen lymphocytes obtained by homogenizing the spleen with asyringe. Typically, a spleen from an immunized mouse containsapproximately 5×10⁷ to 2×10⁸ lymphocytes.

The antibody producing B lymphocytes from the immunized animal are thenfused with cells of an immortal myeloma cell, generally one of the samespecies as the animal that was immunized. Myeloma cell lines suited foruse in hybridoma producing fusion procedures preferably are non antibodyproducing, have high fusion efficiency, and enzyme deficiencies thatrender then incapable of growing in certain selective media whichsupport the growth of only the desired fused cells (hybridomas).

Any one of a number of myeloma cells may be used, as are known to thoseof skill in the art (Goding, pp. 65 66, 1986; Campbell, pp. 75 83,1984). cites). For example, where the immunized animal is a mouse, onemay use P3 X63/Ag8, X63 Ag8.653, NS1/1.Ag 41, Sp210 Ag14, FO, NSO/U, MPC11, MPC11 X45 GTG 1.7 and S194/5XX0 Bul; for rats, one may useR210.RCY3, Y3 Ag 1.2.3, IR983F and 4B210; and U 266, GM1500 GRG2, LICRLON HMy2 and UC729 6 are all useful in connection with human cellfusions. See Yoo et al. (2002), for a discussion of myeloma expressionsystems.

One murine myeloma cell is the NS-1 myeloma cell line (also termedP3-NS-1-Ag4-1), which is readily available from the NIGMS Human GeneticMutant Cell Repository by requesting cell line repository number GM3573.Another mouse myeloma cell line that may be used is the 8 azaguanineresistant mouse murine myeloma SP2/0 non producer cell line.

Methods for generating hybrids of antibody producing spleen or lymphnode cells and myeloma cells usually comprise mixing somatic cells withmyeloma cells in a 2:1 proportion, though the proportion may vary fromabout 20:1 to about 1:1, respectively, in the presence of an agent oragents (chemical or electrical) that promote the fusion of cellmembranes. Fusion methods using Sendai virus have been described byKohler and Milstein (1975; 1976), and those using polyethylene glycol(PEG), such as 37% (v/v) PEG, by Gefter et al., (1977). The use ofelectrically induced fusion methods is also appropriate (Goding pp. 7174, 1986).

Fusion procedures usually produce viable hybrids at low frequencies,about 1×10⁻⁶ to 1×10⁻⁸. However, this does not pose a problem, as theviable, fused hybrids are differentiated from the parental, unfusedcells (particularly the unfused myeloma cells that would normallycontinue to divide indefinitely) by culturing in a selective medium. Theselective medium is generally one that contains an agent that blocks thede novo synthesis of nucleotides in the tissue culture media. Exemplaryand preferred agents are aminopterin, methotrexate, and azaserine.Aminopterin and methotrexate block de novo synthesis of both purines andpyrimidines, whereas azaserine blocks only purine synthesis. Whereaminopterin or methotrexate is used, the media is supplemented withhypoxanthine and thymidine as a source of nucleotides (HAT medium).Where azaserine is used, the media is supplemented with hypoxanthine.

The preferred selection medium is HAT. Only cells capable of operatingnucleotide salvage pathways are able to survive in HAT medium. Themyeloma cells are defective in key enzymes of the salvage pathway, e.g.,hypoxanthine phosphoribosyl transferase (HPRT), and they cannot survive.The B cells can operate this pathway, but they have a limited life spanin culture and generally die within about two weeks. Therefore, the onlycells that can survive in the selective media are those hybrids formedfrom myeloma and B cells.

This culturing provides a population of hybridomas from which specifichybridomas are selected. Typically, selection of hybridomas is performedby culturing the cells by single-clone dilution in microtiter plates,followed by testing the individual clonal supernatants (after about twoto three weeks) for the desired reactivity. The assay should besensitive, simple and rapid, such as radioimmunoassays, enzymeimmunoassays, cytotoxicity assays, plaque assays, dot immunobindingassays, and the like.

The selected hybridomas would then be serially diluted and cloned intoindividual antibody producing cell lines, which clones can then bepropagated indefinitely to provide MAbs. The cell lines may be exploitedfor MAb production in two basic ways. First, a sample of the hybridomacan be injected (often into the peritoneal cavity) into ahistocompatible animal of the type that was used to provide the somaticand myeloma cells for the original fusion (e.g., a syngeneic mouse).Optionally, the animals are primed with a hydrocarbon, especially oilssuch as pristane (tetramethylpentadecane) prior to injection. Theinjected animal develops tumors secreting the specific monoclonalantibody produced by the fused cell hybrid. The body fluids of theanimal, such as serum or ascites fluid, can then be tapped to provideMAbs in high concentration. Second, the individual cell lines could becultured in vitro, where the MAbs are naturally secreted into theculture medium from which they can be readily obtained in highconcentrations.

Further, expression of antibodies (or other moieties therefrom) fromproduction cell lines can be enhanced using a number of knowntechniques. For example, the glutamine synthetase and DHFR geneexpression systems are common approaches for enhancing expression undercertain conditions. High expressing cell clones can be identified usingconventional techniques, such as limited dilution cloning and Microdroptechnology. The GS system is discussed in whole or part in connectionwith European Patent Nos. 0 216 846, 0 256 055, and 0 323 997 andEuropean Patent Application No. 89303964.4.

MAbs produced by either means may be further purified, if desired, usingfiltration, centrifugation and various chromatographic methods such asHPLC or affinity chromatography. Fragments of the monoclonal antibodiescan be obtained from the monoclonal antibodies so produced by methodswhich include digestion with enzymes, such as pepsin or papain, and/orby cleavage of disulfide bonds by chemical reduction. Alternatively,monoclonal antibody fragments can be synthesized using an automatedpeptide synthesizer.

It is also contemplated that a molecular cloning approach may be used togenerate monoclonal antibodies. In one embodiment, combinatorialimmunoglobulin phagemid libraries are prepared from RNA isolated fromthe spleen of the immunized animal, and phagemids expressing appropriateantibodies are selected by panning using cells expressing the antigenand control cells. The advantages of this approach over conventionalhybridoma techniques are that approximately 10⁴ times as many antibodiescan be produced and screened in a single round, and that newspecificities are generated by H and L chain combination which furtherincreases the chance of finding appropriate antibodies.

Another embodiment concerns producing antibodies, for example, as isfound in U.S. Pat. No. 6,091,001, which describes methods to produce acell expressing an antibody from a genomic sequence of the cellcomprising a modified immunoglobulin locus using Cre-mediatedsite-specific recombination is disclosed. The method involves firsttransfecting an antibody-producing cell with a homology-targeting vectorcomprising a lox site and a targeting sequence homologous to a first DNAsequence adjacent to the region of the immunoglobulin loci of thegenomic sequence which is to be converted to a modified region, so thefirst lox site is inserted into the genomic sequence via site-specifichomologous recombination. Then the cell is transfected with alox-targeting vector comprising a second lox site suitable forCre-mediated recombination with the integrated lox site and a modifyingsequence to convert the region of the immunoglobulin loci to themodified region. This conversion is performed by interacting the loxsites with Cre in vivo, so that the modifying sequence inserts into thegenomic sequence via Cre-mediated site-specific recombination of the loxsites.

Alternatively, monoclonal antibody fragments can be synthesized using anautomated peptide synthesizer, or by expression of full-length gene orof gene fragments in E. coli.

2. Antibody and Polypeptide Conjugates

Embodiments provide antibodies and antibody-like molecules against aT-cell anergy gene product of Table 2 or against a T-cell anergy relatedcell surface receptor selected from Semaphorin 7A (Sema7A),Class-I-MHC-restricted T cell associated molecule (Crtam),lymphocyte-activation gene 3 (Lag3), tumor necrosis factor receptorsuperfamily member 9 (Tnfrsf9, also known as 4-1BB), Neuritin (Nrn1),CLIP (CD74), Tnfsf11 (RANKL, CD254), prostaglandin F2 receptor inhibitor(Ptgfrn, CD9-P1, CD315) or oxidized low density lipoprotein receptor 1(LOX1, OLR1) protein, polypeptide or peptide that are linked to at leastone agent to form an antibody conjugate or payload. In order to increasethe efficacy of antibody molecules as diagnostic or therapeutic agents,it is conventional to link or covalently bind or complex at least onedesired molecule or moiety. Such a molecule or moiety may be, but is notlimited to, at least one effector or reporter molecule. Effectormolecules comprise molecules having a desired activity, e.g., cytotoxicactivity. Non-limiting examples of effector molecules which have beenattached to antibodies include toxins, therapeutic enzymes, antibiotics,radio-labeled nucleotides and the like. By contrast, a reporter moleculeis defined as any moiety which may be detected using an assay.Non-limiting examples of reporter molecules which have been conjugatedto antibodies include enzymes, radiolabels, haptens, fluorescent labels,phosphorescent molecules, chemiluminescent molecules, chromophores,luminescent molecules, photoaffinity molecules, colored particles orligands, such as biotin.

Certain examples of antibody conjugates are those conjugates in whichthe antibody is linked to a detectable label. “Detectable labels” arecompounds and/or elements that can be detected due to their specificfunctional properties, and/or chemical characteristics, the use of whichallows the antibody to which they are attached to be detected, and/orfurther quantified if desired.

Antibody conjugates are generally preferred for use as diagnosticagents. Antibody diagnostics generally fall within two classes, thosefor use in in vitro diagnostics, such as in a variety of immunoassays,and/or those for use in vivo diagnostic protocols, generally known as“antibody directed imaging”. Many appropriate imaging agents are knownin the art, as are methods for their attachment to antibodies (see, fore.g., U.S. Pat. Nos. 5,021,236; 4,938,948; and 4,472,509, eachincorporated herein by reference). The imaging moieties used can beparamagnetic ions; radioactive isotopes; fluorochromes; NMR-detectablesubstances; X-ray imaging.

In the case of paramagnetic ions, one might mention by way of exampleions such as chromium (III), manganese (II), iron (III), iron (II),cobalt (II), nickel (II), copper (II), neodymium (III), samarium (III),ytterbium (III), gadolinium (III), vanadium (II), terbium (III),dysprosium (III), holmium (III) and/or erbium (III), with gadoliniumbeing particularly preferred. Ions useful in other contexts, such asX-ray imaging, include but are not limited to lanthanum (III), gold(III), lead (II), and especially bismuth (III).

In the case of radioactive isotopes for therapeutic and/or diagnosticapplication, one might use astatine²¹¹, ¹⁴carbon, ⁵¹chromium,³⁶chlorine, ⁵⁷cobalt, ⁵⁸cobalt, copper⁶⁷, ¹⁵²Eu, gallium⁶⁷, ³hydrogen,iodine¹²³, iodine¹²⁵, iodine¹³¹, indium¹¹¹, ⁵⁹iron, ³²phosphorus,rhenium¹⁸⁶, rhenium¹⁸⁸, ⁷⁵selenium, ³⁵sulphur, technicium^(99m) and/oryttrium⁹⁰. ¹²⁵I is often used in certain embodiments, andtechnicium^(99m) and/or indium¹¹¹ are also often used due to their lowenergy and suitability for long range detection. Radioactively labeledmonoclonal antibodies may be produced according to well-known methods inthe art. For instance, monoclonal antibodies can be iodinated by contactwith sodium and/or potassium iodide and a chemical oxidizing agent suchas sodium hypochlorite, or an enzymatic oxidizing agent, such aslactoperoxidase. Monoclonal antibodies may be labeled with technetium99mby ligand exchange process, for example, by reducing pertechnate withstannous solution, chelating the reduced technetium onto a Sephadexcolumn and applying the antibody to this column. Alternatively, directlabeling techniques may be used, e.g., by incubating pertechnate, areducing agent such as SNCl₂, a buffer solution such as sodium-potassiumphthalate solution, and the antibody. Intermediary functional groupswhich are often used to bind radioisotopes which exist as metallic ionsto antibody are diethylenetriaminepentaacetic acid (DTPA) or ethylenediaminetetracetic acid (EDTA).

Among the fluorescent labels contemplated for use as conjugates includeAlexa 350, Alexa 430, AMCA, BODIPY 630/650, BODIPY 650/665, BODIPY-FL,BODIPY-R6G, BODIPY-TMR, BODIPY-TRX, Cascade Blue, Cy3, Cy5,6-FAM,Fluorescein Isothiocyanate, HEX, 6-JOE, Oregon Green 488, Oregon Green500, Oregon Green 514, Pacific Blue, REG, Rhodamine Green, RhodamineRed, Renographin, ROX, TAMRA, TET, Tetramethylrhodamine, and/or TexasRed, among others.

Antibody conjugates include those intended primarily for use in vitro,where the antibody is linked to a secondary binding ligand and/or to anenzyme (an enzyme tag) that will generate a colored product upon contactwith a chromogenic substrate. Examples of suitable enzymes include, butare not limited to, urease, alkaline phosphatase, (horseradish) hydrogenperoxidase or glucose oxidase. Preferred secondary binding ligands arebiotin and/or avidin and streptavidin compounds. The use of such labelsis well known to those of skill in the art and are described, forexample, in U.S. Pat. Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345;4,277,437; 4,275,149 and 4,366,241; each incorporated herein byreference.

Yet another known method of site-specific attachment of molecules toantibodies comprises the reaction of antibodies with hapten-basedaffinity labels. Essentially, hapten-based affinity labels react withamino acids in the antigen binding site, thereby destroying this siteand blocking specific antigen reaction. However, this may not beadvantageous since it results in loss of antigen binding by the antibodyconjugate.

Molecules containing azido groups may also be used to form covalentbonds to proteins through reactive nitrene intermediates that aregenerated by low intensity ultraviolet light (Potter & Haley, 1983). Inparticular, 2- and 8-azido analogues of purine nucleotides have beenused as site-directed photoprobes to identify nucleotide bindingproteins in crude cell extracts (Owens & Haley, 1987; Atherton et al.,1985). The 2- and 8-azido nucleotides have also been used to mapnucleotide binding domains of purified proteins (Khatoon et al., 1989;King et al., 1989; and Dholakia et al., 1989) and may be used asantibody binding agents.

Several methods are known in the art for the attachment or conjugationof an antibody to its conjugate moiety. Some attachment methods involvethe use of a metal chelate complex employing, for example, an organicchelating agent such a diethylenetriaminepentaacetic acid anhydride(DTPA); ethylenetriaminetetraacetic acid; N-chloro-p-toluenesulfonamide;and/or 1,3,4,6-tetrachloro-3a,6a-diphenyl-glycouril attached to theantibody (U.S. Pat. Nos. 4,472,509 and 4,938,948, each incorporatedherein by reference). Monoclonal antibodies may also be reacted with anenzyme in the presence of a coupling agent such as glutaraldehyde orperiodate. Conjugates with fluorescein markers are prepared in thepresence of these coupling agents or by reaction with an isothiocyanate.In U.S. Pat. No. 4,938,948, imaging of breast tumors is achieved usingmonoclonal antibodies and the detectable imaging moieties are bound tothe antibody using linkers such as methyl-p-hydroxybenzimidate orN-succinimidyl-3-(4-hydroxyphenyl)propionate.

In some embodiments, derivatization of immunoglobulins by selectivelyintroducing sulfhydryl groups in the Fc region of an immunoglobulin,using reaction conditions that do not alter the antibody combining siteare contemplated. Antibody conjugates produced according to thismethodology are disclosed to exhibit improved longevity, specificity andsensitivity (U.S. Pat. No. 5,196,066, incorporated herein by reference).Site-specific attachment of effector or reporter molecules, wherein thereporter or effector molecule is conjugated to a carbohydrate residue inthe Fc region have also been disclosed in the literature (O'Shannessy etal., 1987). This approach has been reported to produce diagnosticallyand therapeutically promising antibodies which are currently in clinicalevaluation.

In some embodiments, antibodies against a T-cell anergy gene product ofTable 2 or a T-cell anergy related cell surface receptor selected fromSemaphorin 7A (Sema7A), Class-I-MHC-restricted T cell associatedmolecule (Crtam), lymphocyte-activation gene 3 (Lag3), tumor necrosisfactor receptor superfamily member 9 (Tnfrsf9, also known as 4-1BB),Neuritin (Nrn1), CLIP (CD74), Tnfsf11 (RANKL, CD254), prostaglandin F2receptor inhibitor (Ptgfrn, CD9-P1, CD315) or oxidized low densitylipoprotein receptor 1 (LOX1, OLR1) are linked to semiconductornanocrystals such as those described in U.S. Pat. Nos. 6,048,616;5,990,479; 5,690,807; 5,505,928; 5,262,357 (all of which areincorporated herein in their entireties); as well as PCT Publication No.99/26299 (published May 27, 1999). In particular, exemplary materialsfor use as semiconductor nanocrystals in the biological and chemicalassays include, but are not limited to, those described above, includinggroup II-VI, III-V and group IV semiconductors such as ZnS, ZnSe, ZnTe,CdS, CdSe, CdTe, MgS, MgSe, MgTe, CaS, CaSe, CaTe, SrS, SrSe, SrTe, BaS,BaSe, BaTe, GaN, GaP, GaAs, GaSb, InP, InAs, InSb, AlS, AlP, AlSb, PbS,PbSe, Ge and Si and ternary and quaternary mixtures thereof. Methods forlinking semiconductor nanocrystals to antibodies are described in U.S.Pat. Nos. 6,630,307 and 6,274,323.

III. Therapeutic Methods

A. Therapeutic siRNA

Small interfering RNA (siRNA), sometimes known as short interfering RNAor silencing RNA, are double-stranded RNA molecules, 20-25 base pairs inlength. siRNAs form part of the RNA interference (RNAi) pathway, wherethey interfere with the expression of specific genes with complementarynucleotide sequence. Any of the embodiments discussed above regardingnucleic acids may be implemented with respect to siRNA molecules.

Therapeutic siRNA may be administered to a patient to modulate theexpression of one or more of the genes identified as involved in T-cellanergy. Therapeutic siRNA may also target the expression of the spectrumof genes/gene products upregulated in an Egr2-dependent fashion. Thedesign of siRNA to target expression of a gene is a process well knownin the art (Nat Biotechnol. 2004 March; 22(3):326-30. Rational siRNAdesign for RNA interference. Reynolds A, Leake D, Boese Q, Scaringe S,Marshall W S, Khvorova A.). Design of siRNA to target any of the genesidentified in T-cell anergy or T-cell hyporesponsiveness may be doneusing the sequences references by Entrez reference number of genes aslisted in FIG. 1C.

The generation of siRNA molecules may, but is not limited to,methodology described in paragraphs above. In addition, the nucleic acidmolecules described above may be employed as siRNA molecules targeting aT cell anergic gene.

Immunotherapy may be used in conjunction with siRNA methods describedabove. Immunotherapy approaches may include ex vivo and in vivoapproaches to increase the immunogenicity of patient tumour cells, suchas transfection with cytokines such as interleukins (such as interleukin2 or interleukin 4), interferons (α, β, γ), or granulocyte macrophagecolony stimulating factor. Immunotherapy approaches may also involveusing transfected immune cells such as cytokine transfected dendriticcells or approaches using cytokine transfected tumour cell lines andusing anti idiotypic antibodies.

Immunotherapy approaches may also include approaches to decrease thefunction of immune suppressive cells such as regulatory T cells,myeloid-derived suppressor cells or IDO (indoleamine2,3,-deoxygenase)-expressing dendritic cells, and approaches usingcancer vaccines consisting of proteins or peptides derived fromtumour-associated antigens such as NY-ESO-1, MAGE-3, WT1 or Her2/neu.

Immunotherapy approaches may also target PD-1 and other molecules whichsignal through interactions with PD-1, such as programmed death ligand 1(PD-L1) and programmed death ligand 2 (PD-L2). The inhibition of PD-L1signaling can be ued as immunotherapeutic means to enhance T cellimmunity for the treatment of cancer (e.g., tumor immunity) andinfection.

B. Antibody Therapy

In certain immunotherapy approaches, a binding polypeptide thatspecifically recognizes and binds a substance, such as anotherpolypeptide may be administered to modulate the activity of one or moreproteins involved in T-cell anergy. In certain instances, the bindingpolypeptide is an antibody or a binding fragment of an antibody. Thebinding polypeptide may comprises at least 1, 2, 3, 4, 5, or 6 CDRs froma monoclonal antibody. Certain types of immunotherapy may compriseantibody therapy that comprises monoclonal antibodies.

Immunotherapy comprising antibody therapy specifically contemplate anantibody that is part of the antibody therapy and binds a T-cell anergygene product of at least one of the T-cell anergy genes listed in Table2. In other specific immunotherapeutic methods the antibody binds acell-surface receptor that is the product of at least one of the T-cellanergy genes listed in Table 2. Some examples include an antibody thatbinds Semaphorin 7A (Sema7A), Class-I-MHC-restricted T cell associatedmolecule (Crtam), lymphocyte-activation gene 3 (Lag3), tumor necrosisfactor receptor superfamily member 9 (Tnfrsf9, also known as 4-1BB),Neuritin (Nrn1), CLIP (CD74), Tnfsf11 (RANKL, CD254), prostaglandin F2receptor inhibitor (Ptgfrn, CD9-P1, CD315) or oxidized low densitylipoprotein receptor 1 (LOX1, OLR1).

Immunotherapy comprising antibody therapy may comprise at least one orat least two antibodies that bind different cell surface receptorsselected from the T-cell anergy genes of Table 2. In additionalembodiments, the antibody therapy comprises at least three, four or fiveantibodies that bind different cell surface receptors selected from theT-cell anergy genes of Table 2.

Antibody therapy can comprise a monoclonal antibody that bindslymphocyte-activation gene 3 (Lag3). A Lag3 antibody may be any of theLag3 antibodies given below or any other Lag3 antibody known orcommercially available. In certain embodiments the anti-Lag3 antibody ismonoclonal clone C9B7W or any humanized or fully human antibodycomprising the CDRs of C9B7W.

Antibody therapy can comprise a monoclonal antibody that binds 4-1BB(also known as tumor necrosis factor receptor superfamily member 9(Tnfrsf9)). A 4-1BB antibody may be any of the 4-1BB antibodies givenbelow or any other 4-1BB antibody known or commercially available. Incertain aspects, the anti-4-1BB antibody is monoclonal antibody cloneLOB12.3. In still other embodiments, the antibody therapy comprises amonoclonal antibody that binds Lag3 and a monoclonal antibody that binds4-1BB. In yet other embodiments, antibody therapy may be administered incombination with other treatments contemplated herein to suppress theactivity or expression of T-cell anergy genes or their products.

1. Lag3 Antibodies

A Lag3 antibody may be any of the Lag3 antibodies given in Table 3Abelow. Specifically, a Lag3 antibody may be any one of monoclonal 17B4,11E3, 1D4B, BMS-986016 or C9B7W or any chimeric, humanized or fullyhuman antibody comprising the CDRs of anti-Lag3 monoclonals 17B4, 11E3,1D4B, BMS-986016 or C9B7W. In certain embodiments the anti-Lag3 antibodyis monoclonal clone C9B7W. In some embodiments, 2, 3, 4, or moredifferent Lag antibodies are used.

TABLE 3A Company Lag3 antibody specificity/type Cat. No. Company LAG3(clone 17B4) human/mouse LS-B2237 LifeSpan monoclonal BioSciences, Inc.LAG3 (clone 11E3) human/mouse LS-C18690 LifeSpan monoclonal BioSciences,Inc. LAG3 mouse/rat LS-C4474 LifeSpan monoclonal BioSciences, Inc. LAG3(aa110-140) human/rabbit LS-C165666 LifeSpan polyclonal BioSciences,Inc. LAG3 (clone 11E3) human/mouse LS-C18690 LifeSpan monoclonalBioSciences, Inc. LAG3 (clone 1D4B) mouse/rat LS-C189811 LifeSpanmonoclonal BioSciences, Inc. LAG3(clone mouse/rat 14-2231-85 affymetrixC9B7W) monoclonal eBioscience Goat Polyclonal FAB2319 R&D Clone 333210Mouse IgG2A MAB2319 R&D Clone 333213 Mouse IgG2A MAB23192 R&D Clone333221 Mouse IgG2B BAM23191 R&D Clone 874501 Mouse IgG1 MAB23193 R&D3DS223H Mouse IgG1 46-2239-41 Ebioscience Clone EPR4392(2) Rabbitab180187) AbCam monoclonal Clone 11E3 Mouse (ab40465) AbCam Monoclonal

A Lag3 antibody may also be any of the Lag3 antibodies disclosed inWO2010/019570, incorporated herein by reference in its entirety. TheseLag3 antibodies include anti-Lag3 (clone 25F7), anti-Lag3 (clone 26H10),anti-Lag3 (clone 25E3), anti-Lag3 (clone 8B7), anti-Lag3 (clone 11F2)and anti-Lag3 (clone 17E5). A Lag3 antibody may also be any chimeric,humanized or fully human antibody comprising the CDRs of anti-Lag3(clone 25F7), anti-Lag3 (clone 26H10), anti-Lag3 (clone 25E3), anti-Lag3(clone 8B7), anti-Lag3 (clone 11F2) and anti-Lag3 (clone 17E5). The CDRsand variable regions of these clones are as indicated in Table 3B below(VH=heavy chain variable region; VK=light chain variable region).

TABLE 3B Antibody SEQ ID NO: Description anti-Lag3 (25F7) 45 VH CDR1 51VH CDR2 57 VH CDR3 63 VK CDR1 69 VK CDR2 75 VK CDR3 81 VH 87 VKanti-Lag3 (26H10) 46 VH CDR1 52 VH CDR2 58 VH CDR3 64 VK CDR1 70 VK CDR276 VK CDR3 82 VH 88 VK anti-Lag3 (25E3) 47 VH CDR1 53 VH CDR2 59 VH CDR365 VK CDR1 71 VK CDR2 77 VK CDR3 83 VH 89 VK anti-Lag3 (8B7) 48 VH CDR154 VH CDR2 60 VH CDR3 66 VK CDR1 72 VK CDR2 78 VK CDR3 84 VH 90 VKanti-Lag3 (11F2) 49 VH CDR1 55 VH CDR2 61 VH CDR3 67 VK CDR1 73 VK CDR279 VK CDR3 85 VH 91 VK anti-Lag3 (17E5) 50 VH CDR1 56 VH CDR2 62 VH CDR368 VK CDR1 74 VK CDR2 80 VK CDR3 86 VH 92 VK

2. 4-1BB Antibodies

A 4-1BB antibody may be any of the 4-1BB antibodies given in Table 4Abelow. Specifically, a 4-1BB antibody may be any one of monoclonalLOB12.3, 4b4-1, ectodomain clone BBK-2, 5H5, BMS-663513 (also known asurelumab) or BBK-2 or any chimeric, humanized or fully human antibodycomprising the CDRs of anti-4-1BB monoclonals LOB12.3, 4b4-1, ectodomainclone BBK-2, 5H5, BMS-663513 (also known as urelumab) or BBK-2.

TABLE 4A Company 4-1BB antibody specificity/type Cat. No. CompanyAnti-4-1BB human/mouse LS-C134760 LifeSpan clone 4b4-1 monoclonalBioSciences, Inc. Anti-4-1BB human/mouse LS-C88297 LifeSpan (ectodomain)monoclonal BioSciences, Inc. clone BBK-2 Anti-4-1BB human/goatLS-C104311 LifeSpan polyclonal BioSciences, Inc. Anti-4-1BB human/mouseLS-C175677 LifeSpan clone 5H5 monoclonal BioSciences, Inc. Anti-4-1BBhuman/mouse LS-C88295 LifeSpan clone BBK-2 monoclonal BioSciences, Inc.Anti-4-1BB Mouse IgG1 12-1379-41 Ebioscience/Many Clone 4B4 Anti-4-1BBmouse IgG1 552532 BD Pharmingen Clone h41BB-M127 Anti-4-1BB Fully humanBristol-Myers BMS-663513 IgG4 Squibb (Urelumab)

A 4-1BB antibody may also be any of the 4-1BB antibodies disclosed inU.S. Pat. No. 8,137,667 and U.S. Pat. No. 8,337,850, the contents ofwhich are incorporated herein by reference in their entirety. These4-1BB antibodies include the anti-4-1BB antibodies (anti-human 4-1-bb,MOR-6032 and MOR-7361) described in Table 4B. A 4-1BB antibody may alsobe any chimeric, humanized or fully human antibody comprising the CDRsof the anti-4-1BB antibodies of Table 4B. The CDRs and variable regionsof these clones are as indicated in the table below (HC=heavy chain;LC=light chain).

TABLE 4B SEQ ID Antibody NO: Description Anti-human 93 CDRs at: 4-1bbamino acid position 50-54 of SEQ ID NO: 93 (fully amino acid position69-84 of SEQ ID NO: 93 human Ab) amino acid position 117-129 of SEQ IDNO: 93 HC at amino acid position 20-467 94 CDRs at: amino acid position44-54 of SEQ ID NO: 94 amino acid position 70-76 of SEQ ID NO: 94 aminoacid position 109-119 of SEQ ID NO: 94 LC at amino acid position 21-236Anti-human 99 Full length heavy chain 4-1bb 104 Full length light chainMOR-6032 98 Variable Region of HC 103 Variable Region of LC 95 H-CDR1 96H-CDR2 97 H-CDR3 100 L-CDR1 101 L-CDR2 102 L-CDR3 Anti-4-1bb 109 Fulllength heavy chain MOR-7361 114 Full length light chain 108 VariableRegion of HC 113 Variable Region of LC 105 H-CDR1 106 H-CDR2 107 H-CDR3110 L-CDR1 111 L-CDR2 112 L-CDR3

C. Combination Therapy

The compositions and related methods, particularly administration of ansiRNA to inhibit expression of T-cell anergy gene or a peptide, antibodyor drug to inhibit said gene product to a patient/subject, may also beused in combination with the administration of traditional therapies orimmunotherapies, such as, for example, traditional cancer therapies(such as chemotherapeutics, radiation, and/or surgery).

The compositions and related methods, particularly administration of ansiRNA to inhibit expression of T-cell anergy gene or a peptide, antibodyor drug to inhibit said gene product to a patient/subject may also beused in combination with the administration of one or more anti-cancerdrugs that include but are not limited to Abiraterone Acetate,Abitrexate (Methotrexate), Abraxane (Paclitaxel Albumin-stabilizedNanoparticle Formulation), ABVD, ABVE, ABVE-PC, AC, AC-T, Adcetris(Brentuximab Vedotin), ADE, Ado-Trastuzumab Emtansine, Adriamycin(Doxorubicin Hydrochloride), Adrucil (Fluorouracil), Afatinib Dimaleate,Afinitor (Everolimus), Aldara (Imiquimod), Aldesleukin, Alemtuzumab,Alimta (Pemetrexed Disodium), Aloxi (Palonosetron Hydrochloride),Ambochlorin (Chlorambucil), Amboclorin (Chlorambucil), AminolevulinicAcid, Anastrozole, Aprepitant, Aredia (Pamidronate Disodium), Arimidex(Anastrozole), Aromasin (Exemestane), Arranon (Nelarabine), ArsenicTrioxide, Arzerra (Ofatumumab), Asparaginase Erwinia chrysanthemi,Avastin (Bevacizumab), Axitinib, Azacitidine, BEACOPP, BendamustineHydrochloride, BEP, Bevacizumab, Bexarotene, Bexxar (Tositumomab and I131 Iodine Tositumomab), Bleomycin, Bortezomib, Bosulif (Bosutinib),Bosutinib, Brentuximab Vedotin, Cabazitaxel, Cabozantinib-S-Malate, CAF,Campath (Alemtuzumab), Camptosar (Irinotecan Hydrochloride),Capecitabine, CAPDX, Carboplatin, CARBOPLATIN-TAXOL, Carfilzomib, CeeNU(Lomustine), Cerubidine (Daunorubicin Hydrochloride), Cervarix(Recombinant HPV Bivalent Vaccine comprising recombinant L1 protein ofHPV types 16 and 18), Cetuximab, Chlorambucil, CHLORAMBUCIL-PREDNISONE,CHOP, Cisplatin, Clafen (Cyclophosphamide), Clofarabine, Clofarex(Clofarabine), Clolar (Clofarabine), CMF, Cometriq(Cabozantinib-S-Malate), COPP, COPP-ABV, Cosmegen (Dactinomycin),Crizotinib, CVP, Cyclophosphamide, Cyfos (Ifosfamide), Cytarabine,Cytarabine, Liposomal, Cytosar-U (Cytarabine), Cytoxan(Cyclophosphamide), Dabrafenib, Dacarbazine, Dacogen (Decitabine),Dactinomycin, Dasatinib, Daunorubicin Hydrochloride, Decitabine,Degarelix, Denileukin Diftitox, Denosumab, DepoCyt (LiposomalCytarabine), DepoFoam (Liposomal Cytarabine), Dexrazoxane Hydrochloride,Docetaxel, Doxil (Doxorubicin Hydrochloride Liposome), DoxorubicinHydrochloride, Doxorubicin Hydrochloride Liposome, Dox-SL (DoxorubicinHydrochloride Liposome), DTIC-Dome (Dacarbazine), Efudex (Fluorouracil),Elitek (Rasburicase), Ellence (Epirubicin Hydrochloride), Eloxatin(Oxaliplatin), Eltrombopag Olamine, Emend (Aprepitant), Enzalutamide,Epirubicin Hydrochloride, EPOCH, Erbitux (Cetuximab), Eribulin Mesylate,Erivedge (Vismodegib), Erlotinib Hydrochloride, Erwinaze (AsparaginaseErwinia chrysanthemi), Etopophos (Etoposide Phosphate), Etoposide,Etoposide Phosphate, Evacet (Doxorubicin Hydrochloride Liposome),Everolimus, Evista (Raloxifene Hydrochloride), Exemestane, Fareston(Toremifene), Faslodex (Fulvestrant), FEC, Femara (Letrozole),Filgrastim, Fludara (Fludarabine Phosphate), Fludarabine Phosphate,Fluoroplex (Fluorouracil), Fluorouracil, Folex (Methotrexate), Folex PFS(Methotrexate), FOLFIRI, FOLFIRI-BEVACIZUMAB, FOLFIRI-CETUXIMAB,FOLFIRINOX, FOLFOX, Folotyn (Pralatrexate), FU-LV, Fulvestrant, Gardasil(Recombinant HPV Quadrivalent Vaccine comprising recombinant L1 proteinof HPV types 6, 11, 16, and 18), Gazyva (Obinutuzumab), Gefitinib,Gemcitabine Hydrochloride, GEMCITABINE-CISPLATIN,GEMCITABINE-OXALIPLATIN, Gemtuzumab Ozogamicin, Gemzar (GemcitabineHydrochloride), Gilotrif (Afatinib Dimaleate), Gleevec (ImatinibMesylate), Glucarpidase, Goserelin Acetate, Halaven (Eribulin Mesylate),Herceptin (Trastuzumab), HPV Bivalent Vaccine, Recombinant, HPVQuadrivalent Vaccine, Recombinant, Hycamtin (Topotecan Hydrochloride),Ibritumomab Tiuxetan, Ibrutinib, ICE, Iclusig (Ponatinib Hydrochloride),Ifex (Ifosfamide), Ifosfamide, Ifosfamidum (Ifosfamide), ImatinibMesylate, Imbruvica (Ibrutinib), Imiquimod, Inlyta (Axitinib), Intron A(Recombinant Interferon Alfa-2b), Iodine 131 Tositumomab andTositumomab, Ipilimumab, Iressa (Gefitinib), Irinotecan Hydrochloride,Istodax (Romidepsin), Ixabepilone, Ixempra (Ixabepilone), Jakafi(Ruxolitinib Phosphate), Jevtana (Cabazitaxel), Kadcyla (Ado-TrastuzumabEmtansine), Keoxifene (Raloxifene Hydrochloride), Kepivance(Palifermin), Kyprolis (Carfilzomib), Lapatinib Ditosylate,Lenalidomide, Letrozole, Leucovorin Calcium, Leukeran (Chlorambucil),Leuprolide Acetate, Levulan (Aminolevulinic Acid), Linfolizin(Chlorambucil), LipoDox (Doxorubicin Hydrochloride Liposome), LiposomalCytarabine, Lomustine, Lupron (Leuprolide Acetate), Lupron Depot(Leuprolide Acetate), Lupron Depot-Ped (Leuprolide Acetate), LupronDepot-3 Month (Leuprolide Acetate), Lupron Depot-4 Month (LeuprolideAcetate), Marqibo (Vincristine Sulfate Liposome), Matulane (ProcarbazineHydrochloride), Mechlorethamine Hydrochloride, Megace (MegestrolAcetate), Megestrol Acetate, Mekinist (Trametinib), Mercaptopurine,Mesna, Mesnex (Mesna), Methazolastone (Temozolomide), Methotrexate,Methotrexate LPF (Methotrexate), Mexate (Methotrexate), Mexate-AQ(Methotrexate), Mitomycin C, Mitozytrex (Mitomycin C), MOPP, Mozobil(Plerixafor), Mustargen (Mechlorethamine Hydrochloride), Mutamycin(Mitomycin C), Mylosar (Azacitidine), Mylotarg (Gemtuzumab Ozogamicin),Nanoparticle Paclitaxel (Paclitaxel Albumin-stabilized NanoparticleFormulation), Navelbine (Vinorelbine Tartrate), Nelarabine, Neosar(Cyclophosphamide), Neupogen (Filgrastim), Nexavar (Sorafenib Tosylate),Nilotinib, Nolvadex (Tamoxifen Citrate), Nplate (Romiplostim),Obinutuzumab, Ofatumumab, Omacetaxine Mepesuccinate, Oncaspar(Pegaspargase), Ontak (Denileukin Diftitox), OEPA, OPPA, Oxaliplatin,Paclitaxel, Paclitaxel Albumin-stabilized Nanoparticle Formulation,Palifermin, Palonosetron Hydrochloride, Pamidronate Disodium,Panitumumab, Paraplat (Carboplatin), Paraplatin (Carboplatin), PazopanibHydrochloride, Pegaspargase, Peginterferon Alfa-2b, PEG-Intron(Peginterferon Alfa-2b), Pemetrexed Disodium, Perjeta (Pertuzumab),Pertuzumab, Platinol (Cisplatin), Platinol-AQ (Cisplatin), Plerixafor,Pomalidomide, Pomalyst (Pomalidomide), Ponatinib Hydrochloride,Pralatrexate, Prednisone, Procarbazine Hydrochloride, Proleukin(Aldesleukin), Prolia (Denosumab), Promacta (Eltrombopag Olamine),Provenge (Sipuleucel-T), Purinethol (Mercaptopurine), Radium 223Dichloride, Raloxifene Hydrochloride, Rasburicase, R-CHOP, R-CVP,Recombinant HPV Bivalent Vaccine, Recombinant HPV Quadrivalent Vaccine,Recombinant Interferon Alfa-2b, Regorafenib, Revlimid (Lenalidomide),Rheumatrex (Methotrexate), Rituxan (Rituximab), Rituximab, Romidepsin,Romiplostim, Rubidomycin (Daunorubicin Hydrochloride), RuxolitinibPhosphate, Sclerosol Intrapleural Aerosol (Talc), Sipuleucel-T,Sorafenib Tosylate, Sprycel (Dasatinib), STANFORD V, Sterile Talc Powder(Talc), Steritalc (Talc), Stivarga (Regorafenib), Sunitinib Malate,Sutent (Sunitinib Malate), Sylatron (Peginterferon Alfa-2b), Synovir(Thalidomide), Synribo (Omacetaxine Mepesuccinate), TAC, Tafinlar(Dabrafenib), Talc, Tamoxifen Citrate, Tarabine PFS (Cytarabine),Tarceva (Erlotinib Hydrochloride), Targretin (Bexarotene), Tasigna(Nilotinib), Taxol (Paclitaxel), Taxotere (Docetaxel), Temodar(Temozolomide), Temozolomide, Temsirolimus, Thalidomide, Thalomid(Thalidomide), Toposar (Etoposide), Topotecan Hydrochloride, Toremifene,Torisel (Temsirolimus), Tositumomab and I 131 Iodine Tositumomab, Totect(Dexrazoxane Hydrochloride), Trametinib, Trastuzumab, Treanda(Bendamustine Hydrochloride), Trisenox (Arsenic Trioxide), Tykerb(Lapatinib Ditosylate), Vandetanib, VAMP, Vectibix (Panitumumab), VeIP,Velban (Vinblastine Sulfate), Velcade (Bortezomib), Velsar (VinblastineSulfate), Vemurafenib, VePesid (Etoposide), Viadur (Leuprolide Acetate),Vidaza (Azacitidine), Vinblastine Sulfate, Vincasar PFS (VincristineSulfate), Vincristine Sulfate, Vincristine Sulfate Liposome, VinorelbineTartrate, Vismodegib, Voraxaze (Glucarpidase), Vorinostat, Votrient(Pazopanib Hydrochloride), Wellcovorin (Leucovorin Calcium), Xalkori(Crizotinib), Xeloda (Capecitabine), XELOX, Xgeva (Denosumab), Xofigo(Radium 223 Dichloride), Xtandi (Enzalutamide), Yervoy (Ipilimumab),Zaltrap (Ziv-Aflibercept), Zelboraf (Vemurafenib), Zevalin (IbritumomabTiuxetan), Zinecard (Dexrazoxane Hydrochloride), Ziv-Aflibercept,Zoladex (Goserelin Acetate), Zoledronic Acid, Zolinza (Vorinostat),Zometa (Zoledronic Acid) and Zytiga (Abiraterone Acetate).

The compositions and related methods, particularly administration of ansiRNA to inhibit expression of T-cell anergy gene or a peptide, antibodyor drug to inhibit said gene product to a patient/subject may also beused in combination with the administration of radiation therapy thatincludes but is not limited to X-rays, gamma rays, and chargedparticles. The radiation may be delivered by a machine outside the body(external-beam radiation therapy), or it may come from radioactivematerial placed in the body near cancer cells (internal radiationtherapy or brachytherapy). Internal radiation therapy may be systemic(e.g. radioactive iodine). External-beam radiation therapy may include,but is not limited to, 3-dimensional conformal radiation therapy(3D-CRT), Intensity-modulated radiation therapy (IMRT), Image-guidedradiation therapy (IGRT), Tomotherapy, Stereotactic radiosurgery (SRS),Stereotactic body radiation therapy (SBRT), Proton therapy or othercharged particle beams (e.g., electron beams). Internal radiationtherapy or brachytherapy may comprise interstitial brachytherapy whichuses a radiation source placed within tumor tissue and may be used todeliver a dose higher than external beam radiation while causing lessdamage to normal tissue. Brachytherapy may be given as a low-dose rateor high-dose rate treatment. In additional embodiments, brachytherapymay be permanent or temporary. Radiation therapy may comprise systemicradiation therapy. Systemic radiation therapy may comprise a swallowedor injected radioactive substance, that includes, but is not limited toany single, multiple or combination dose of Radioactive iodine (¹³¹I),ibritumomab tiuxetan (Zevalin®), 131 tositumomab (Bexxar®),samarium-153-lexidronam (Quadramet®) and strontium-89 chloride(Metastron®) or any monoclonal bound to a radioactive substance. Thedose of radiation according to different embodiments may be tailored tothe specific disease, condition or cancered being treated. In someembodiments, the single or total dose may be 1-10 gray(Gy), 10-20 Gy,20-40 Gy, 40-60 Gy, or 60-80 Gy, or any value or rage derivable therein.In some embodiments, radiation therapy or dose may be fractionated. Inone embodiment, a total dose may be fractionated per day or per week. Incertain embodiments the daily fractionated dose may be 1.8-2 Gy. It iscontemplated that a total dose may be fractionated into daily or weeklydoses in the range of 0.1 Gy to 10 Gy.

In one aspect, it is contemplated that a therapy is used in conjunctionwith anticancer treatment. Alternatively, the therapy may precede orfollow the other agent treatment by intervals ranging from minutes toweeks. In embodiments where the other treatments and/or a proteins orpolynucleotides are administered separately, one would generally ensurethat a significant period of time did not expire between the time ofeach delivery, such that the therapeutic composition would still be ableto exert an advantageously combined effect on the subject. In suchinstances, it is contemplated that one may administer both modalitieswithin about 12-24 h of each other and, more preferably, within about6-12 h of each other. In some situations, it may be desirable to extendthe time period for administration significantly, however, where severaldays (2, 3, 4, 5, 6 or 7) to several weeks (1, 2, 3, 4, 5, 6, 7 or 8)lapse between the respective administrations.

Various combinations of therapy may be employed, for example anticancertherapy or immunotheraphy is “A” and an siRNA that targets T-cell anergyrelated gene is “B”:

A/B/A B/A/B B/B/A A/A/B A/B/B B/A/A A/B/B/BB/A/B/B B/B/B/A B/B/A/B A/A/B/B A/B/A/B A/B/B/AB/B/A/A B/A/B/A B/A/A/B A/A/A/B B/A/A/A A/B/A/A A/A/B/A

Administration of the antibody compositions to a patient/subject willfollow general protocols for the administration of such compounds,taking into account the toxicity, if any, of the composition. It isexpected that the treatment cycles would be repeated as necessary. It isalso contemplated that various standard therapies, such as hydration,may be applied in combination with the described therapy.

D. General Pharmaceutical Compositions

In some embodiments, pharmaceutical compositions are administered to asubject. Different aspects may involve administering an effective amountof a composition to a subject. In some embodiments, a therapeutic siRNAmay be administered to the patient to alter the expression of genesassociated with T-cell anergy. Alternatively, an expression vectorencoding one or more such siRNAs may be given to a patient as apreventative treatment. Additionally, such compositions can beadministered in combination with traditional therapy or immunotherapy,such as a traditional cancer therapy. Such compositions will generallybe dissolved or dispersed in a pharmaceutically acceptable carrier oraqueous medium.

The phrases “pharmaceutically acceptable” or “pharmacologicallyacceptable” refer to molecular entities and compositions that do notproduce an adverse, allergic, or other untoward reaction whenadministered to an animal or human. As used herein, “pharmaceuticallyacceptable carrier” includes any and all solvents, dispersion media,coatings, antibacterial and antifungal agents, isotonic and absorptiondelaying agents, and the like. The use of such media and agents forpharmaceutical active substances is well known in the art. Exceptinsofar as any conventional media or agent is incompatible with theactive ingredients, its use in immunogenic and therapeutic compositionsis contemplated. Supplementary active ingredients, such as otheranti-infective agents and vaccines, can also be incorporated into thecompositions.

The active compounds can be formulated for parenteral administration,e.g., formulated for injection via the intravenous, intramuscular,sub-cutaneous, or even intraperitoneal routes. Typically, suchcompositions can be prepared as either liquid solutions or suspensions;solid forms suitable for use to prepare solutions or suspensions uponthe addition of a liquid prior to injection can also be prepared; and,the preparations can also be emulsified.

The pharmaceutical forms suitable for injectable use include sterileaqueous solutions or dispersions; formulations including sesame oil,peanut oil, or aqueous propylene glycol; and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersions. In all cases the form must be sterile and must be fluid tothe extent that it may be easily injected. It also should be stableunder the conditions of manufacture and storage and must be preservedagainst the contaminating action of microorganisms, such as bacteria andfungi.

The proteinaceous compositions may be formulated into a neutral or saltform. Pharmaceutically acceptable salts, include the acid addition salts(formed with the free amino groups of the protein) and which are formedwith inorganic acids such as, for example, hydrochloric or phosphoricacids, or such organic acids as acetic, oxalic, tartaric, mandelic, andthe like. Salts formed with the free carboxyl groups can also be derivedfrom inorganic bases such as, for example, sodium, potassium, ammonium,calcium, or ferric hydroxides, and such organic bases as isopropylamine,trimethylamine, histidine, procaine and the like.

A pharmaceutical composition can include a solvent or dispersion mediumcontaining, for example, water, ethanol, polyol (for example, glycerol,propylene glycol, and liquid polyethylene glycol, and the like),suitable mixtures thereof, and vegetable oils. The proper fluidity canbe maintained, for example, by the use of a coating, such as lecithin,by the maintenance of the required particle size in the case ofdispersion, and by the use of surfactants. The prevention of the actionof microorganisms can be brought about by various antibacterial andantifungal agents, for example, parabens, chlorobutanol, phenol, sorbicacid, thimerosal, and the like. In many cases, it will be preferable toinclude isotonic agents, for example, sugars or sodium chloride.Prolonged absorption of the injectable compositions can be brought aboutby the use in the compositions of agents delaying absorption, forexample, aluminum monostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the activecompounds in the required amount in the appropriate solvent with variousof the other ingredients enumerated above, as required, followed byfiltered sterilization or an equivalent procedure. Generally,dispersions are prepared by incorporating the various sterilized activeingredients into a sterile vehicle which contains the basic dispersionmedium and the required other ingredients from those enumerated above.In the case of sterile powders for the preparation of sterile injectablesolutions, the preferred methods of preparation are vacuum-drying andfreeze-drying techniques, which yield a powder of the active ingredient,plus any additional desired ingredient from a previouslysterile-filtered solution thereof.

Administration of the compositions will typically be via any commonroute. This includes, but is not limited to oral, nasal, or buccaladministration. Alternatively, administration may be by orthotopic,intradermal, subcutaneous, intramuscular, intraperitoneal, intranasal,or intravenous injection. In certain embodiments, a vaccine compositionmay be inhaled (e.g., U.S. Pat. No. 6,651,655, which is specificallyincorporated by reference). Such compositions would normally beadministered as pharmaceutically acceptable compositions that includephysiologically acceptable carriers, buffers or other excipients.

An effective amount of therapeutic or prophylactic composition isdetermined based on the intended goal. The term “unit dose” or “dosage”refers to physically discrete units suitable for use in a subject, eachunit containing a predetermined quantity of the composition calculatedto produce the desired responses discussed above in association with itsadministration, i.e., the appropriate route and regimen. The quantity tobe administered, both according to number of treatments and unit dose,depends on the protection desired.

Precise amounts of the composition also depend on the judgment of thepractitioner and are peculiar to each individual. Factors affecting doseinclude physical and clinical state of the subject, route ofadministration, intended goal of treatment (alleviation of symptomsversus cure), and potency, stability, and toxicity of the particularcomposition.

Upon formulation, solutions will be administered in a manner compatiblewith the dosage formulation and in such amount as is therapeutically orprophylactically effective. The formulations are easily administered ina variety of dosage forms, such as the type of injectable solutionsdescribed above.

IV. Examples

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples which follow representtechniques discovered by the inventor to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention.

Example 1—Methods and Materials

Mice and T Cell Clones—

CAR Tg mice expressing the extracellular domain of CAR under control ofa Lck promoter/CD2 enhancer were generated as previously described (Wanet al., 2000). All mice were housed in pathogen-free conditions at theUniversity of Chicago, and all animal protocols were approved by theInstitutional Animal Care and Use Committee. To generate CARTg×Egr2^(flox/flox) Th1 clones, CAR Tg×Egr2^(flox/flox) mice wereimmunized in the hind footpads with chicken ovalbumin (OVA; A5503,Sigma) emulsified in complete Freund's adjuvant (F5881, Sigma). Sevendays later, the draining lymph nodes were harvested, and CD4⁺ Th1 cellclones were derived and maintained as previously described (Fitch etal., 2006; Gajewski and Fitch, 1990).

Adenovirus Transduction—

A Cre-expressing adenovirus was produced as described (Zha et al., 2006;Zha et al., 2008). For T cell transduction, cells were suspended at highdensity of 10×10⁶/mL in DMEM with 2% FBS, incubated with an EV or theCre adenovirus at 37° C. for 50 minutes, transferred to DMEM with 10%FBS, and cultured for another 16 hours at low density of 1×10⁶/mL.

Anergy Induction In Vitro—

In vitro anergy was induced by treating cells overnight with immobilizedanti-CD3 mAb (1 μg/mL; 145-2C11, BioXCell). The cells were thenharvested, washed, and rested for 1-2 days prior to analysis.

ChIP-seq Analysis—

100 ng of DNA from the ChIP assays was ligated to the Illumina adaptoroligo mix according to the manufacturer's protocols. After 16 cycles ofPCR amplification, the product was separated on 2% agarose gels, and DNAfragments between 150-400 by were purified using a Gel Purification Kit(Qiagen). 8 pmoles of DNA was sequenced on an Illumina GAII sequenceraccording to standard protocols. For data analysis, images obtained fromthe sequencer were processed by Illumina image extraction pipelinesoftware. Eland Extended was used to align sequences to mouse genome(NCBI 37/mm9). Non-unique sequences that aligned to more than twodifferent locations were discarded prior to subsequent analysis. QuEST(Valouev et al., 2008) was used to identify enriched binding regions orpeaks. MEME (Bailey et al., 2009) was used for motif identification. Twoindependent ChIP-seq experiments were performed, and genes present inboth datasets were considered as positive.

Gene Expression Profiling Analysis—

All RNA samples used for gene array analysis had RNA IntegrityNumber>8.0, OD260/280 and OD260/230 ratio>1.8. The RNA was labeled,fragmented and hybridized to Affymetrix mouse genome 430 2.0 expressionarrays at the Functional Genomics Core Facility of the University ofChicago (Chicago, Ill.). The arrays were then scanned and CEL intensityfiles were generated by MicroArray Suite 5.0. The gene array analysiswas performed three times using three sets of independently manipulatedsamples. Results from the three gene arrays were combined and analyzedusing dChip software. Specifically, the genes scored as “absent” or withsignal intensity<100 were first filtered out. Among the remaining genes,those with greater than or equal to a 2-fold increase in expression uponanergy induction were considered as anergy-associated genes, and thosewith more than a 1.5-fold reduction upon Egr2 deletion were consideredas Egr2-dependent genes. Genes were considered as positive when theaverage fold changes of the three sets of samples met the thresholdslisted above.

ChIP Assay—

ChIP assays were conducted following the manufacturer's protocol(Millipore). Briefly, 2.5×10⁶ cells were lysed in 500 μL SDS lysisbuffer, and cellular DNA was sheared 6 times with a 15-second pulse plus60-second rest using a Misonix Sonicator 3000 (Qsonica). Forimmunoprecipitation, 200 μL cell lysate supernatant (corresponding to1×10⁶ cells) was diluted 5 fold in ChIP dilution buffer, and anti-Egr2Ab was added at a 1:100 dilution (PRB-236P, Covance). SYBR Green qRT-PCRwas conducted using primers specific for CCL1 intron (forward5′-AATGGCCACATGGAAAACTC-3′ [SEQ ID NO. 1], reverse5′-CCAAACATACCTCGAATACGC-3′ [SEQ ID NO. 2]); Crtam intron (forward5′-TCTGGACAGGAGGGGATGT-3′ [SEQ ID NO. 3], reverse5′-AGGAAACACCCACAGCAAAG-3′ [SEQ ID NO. 4]); Sema7A core promoter(forward 5′-GCTTCTGCTGGTGTTCTGG-3′ [SEQ ID NO. 5], reverse5′-CGCCTACCTTTCCAGACG-3′ [SEQ ID NO. 6]); Lag3 core promoter (forward5′-CTCCAGACCCAGTCCTTCTG-3′ [SEQ ID NO. 7], reverse5′-ACACTTTCCACTGCGAAGC-3′ [SEQ ID NO. 8]); 4-1BB 5′UTR (forward5′-AATCTCTTAACTCAGGAGAGACGTG-3′ [SEQ ID NO. 9], reverse5′-TTCCCACCACAGTGACATTC-3′ [SEQ ID NO. 10]); Nrgn intron (forward5′-GGCTTGGCTCAGATCAGG-3′ [SEQ ID NO. 11], reverse5′-GGGAAAGAATGGTGCTGAAA-3′ [SEQ ID NO. 12]); Nrn1 proximal promoter(forward 5′-GTGACTGATTTTCATCCCAGTG-3′ [SEQ ID NO. 13], reverse5′-ACCAGGACTCCCCGTCTC-3′ [SEQ ID NO. 14]); Bcl2111 core promoter(forward 5′-TCCACTTGGATTCACACCAC-3′ [SEQ ID NO. 15], reverse5′-CAGACATTGGGTGGACGAG-3′ [SEQ ID NO. 16]); Crabp2 proximal promoter(forward 5′-CTTGCCTTCTGACGCTTCTC-3′ [SEQ ID NO. 17], reverse5′-GGGTTCTCCAAGAGCCAAG-3′ [SEQ ID NO. 18]). Primers specific for GJA5were used as controls (forward 5′-ACCATGGAGGTGGCCTTCA-3′ [SEQ ID NO.19], reverse 5′-CATGCAGGGTATCCAGGAAGA-3′ [SEQ ID NO. 20]).

qRT-PCR—

The primers and probes were purchased from IDT, Roche, and AppliedBiosystems. qRT-PCR used primers and probes specific for CCL1 (forward5′-TCACCATGAAACCCACTGC-3′ [SEQ ID NO. 21], reverse5′-AGCAGCAGCTATTGGAGACC-3′ [SEQ ID NO. 22], CTGGCTGC [SEQ ID NO. 23]);Crtam (forward 5′-AGATCCAACAACGAGGAGACA-3′ [SEQ ID NO. 24], reverse5′-TCATGCAACGCTTAGACTGG-3′ [SEQ ID NO. 25], CTGGCTGC [SEQ ID NO. 23]);Sema7A (forward 5′-TCAATCGGCTGCAAGATGT-3′ [SEQ ID NO. 26], reverse5′-CGCAGACAGCTGAGTAGTTCC-3′ [SEQ ID NO. 27], GAGCAGGA [SEQ ID NO. 28]);Lag3 (forward 5′-TGCTTTGGGAAGCTCCAGT-3′ [SEQ ID NO. 29], reverse5′-GCTGCAGGGAAGATGGAC-3′ [SEQ ID NO. 30], CCAGGAGG [SEQ ID NO. 31]);4-1BB (forward 5′-GAACGGTACTGGCGTCTGTC-3′ [SEQ ID NO. 32], reverse5′-CCGGTCTTAAGCACAGACCT-3′ [SEQ ID NO. 33], CTGCTCTC [SEQ ID NO. 34]);Nrgn (forward 5′-AACACCGGCAATGGACTG-3′ [SEQ ID NO. 35], reverse5′-AAACTCGCCTGGATTTTGG-3′ [SEQ ID NO. 36], GCTGGATG [SEQ ID NO. 37]);Nrn (forward 5′-TCCTCGCGGTGCAAATAG-3′ [SEQ ID NO. 38], reverse5′-GCCCTTAAAGACTGCATCACA-3′ [SEQ ID NO. 39], CTGCTCTC [SEQ ID NO. 34]);Bcl2111 (forward 5′-GGAGACGAGTTCAACGAAACTT-3′ [SEQ ID NO. 40], reverse5′-AACAGTTGTAAGATAACCATTTGAGG-3′ [SEQ ID NO. 41], GGCTGAAG [SEQ ID NO.42]); Crabp2 (forward 5′-AAATGGTGTGCGAGCAGAG-3′ [SEQ ID NO. 43], reverse5′-AACGTCATCTGCTGTCATTGTC-3′ [SEQ ID NO. 44], CCAGGAGG [SEQ ID NO. 31]).Relative RNA abundance was determined based on control 18S RNA(Hs99999901_s1, Applied Biosystems).

Immunoblot Analysis—

Equal numbers of T cells were resuspended in ice-cold lysis buffercontaining 50 mM Tris (pH 7.6), 5 mM EDTA, 150 mM NaCl, 0.5% Tritonx-100, 1 mM PMSF, 10 mM NaF, 1 mM Na₃VO₄, and 1× protease inhibitormixture (Roche). After 30-minute incubation on ice, the cells were spunfor 10 minutes at top speed at 4° C., and supernatant was collected. Thecellular lysate was loaded into 10% Tris-HCL gels (Bio-RadLaboratories), separated by SDS-PAGE, and transferred to PVDF membranes(Millipore). Proteins were detected using primary antibodies againstSema7A (1:1000, AF1835, R&D System) and Crabp2 (1:1000, MAB5488,Millipore); secondary antibodies were HRP-linked anti-mouse IgG (1:3000,GE Healthcare). Detection was performed using an ECL Detection Kit (GEHealthcare).

Flow Cytometry—

To stain Crtam, cells were incubated with an anti-Crtam mouse IgG2a (30ng/mL, 10 μl per 1×10⁶ cells at 4° C. for 20 minutes, and then an AlexaFluor 647 goat anti-mouse IgG2a (1:100; A21241, Invitrogen) at 4° C. foranother 20 minutes.

Example 2—Identification of Egr2 Transcriptome Using Microarray-BasedGene Expression Profiling and ChIP-Seq Analyses

Th1 T cell clones anergized by immobilized anti-CD3 were used as theinventors' T cell anergy model. This model has been well-characterizedand can provide sufficient cellular material for microarray and ChIP-seqanalyses (Schwartz, 2003). T cell-specific Egr2 deletion was mediated byuse of a Cre-expressing adenovirus and a Coxsackie/adenovirus receptor(CAR) Tg×Egr2^(flox/flox) mouse in which CAR is expressed exclusively inthe T cell compartment from a Lck promoter/CD2 enhancer cassette. Thissystem allows for peripheral deletion of Egr2 without affecting T celldevelopment in the thymus, as the inventors recently described (Zha etal., 2008). Briefly, OVA-specific Th1 cell clones were generated fromCAR Tg×Egr2^(flox/flox) mice (Fitch et al., 2006; Zha et al., 2008).Egr2 deletion was then achieved by transduction of the CARTg×Egr2^(flox/flox) Th1 T cell clones with the Cre adenovirus. Thissystem was proven very efficient, qRT-PCT and immunoblot confirmedanti-CD3 induced Egr2 expression was decreased to minimal levels afterthe Cre adenovirus mediated Egr2 deletion (Zheng et al, 2012).

In order to map the complete Egr2 transcriptome of anergic T cells, theinventors conducted two sets of genome-wide screens. The first was amicroarray-based gene expression profiling analysis in which anergic Th1cells were compared with or without prior Egr2 deletion. Specifically,CAR Tg×Egr2^(flox/flox) Th1 T cells were infected with an empty (EV) ora Cre-expressing adenovirus. Upon confirmation of Egr2 deletion byimmunoblot, the T cells were anergized by immobilized anti-CD3 for 16hours, and microarray was conducted after 1 days of rest in culturemedium. This analysis helped to identify the set of genes upregulatedupon anergy induction in an Egr2-dependent manner (Supplementary Table1). Gene array analysis revealed that 938 out of the total 45101 probesdemonstrated at least a 2-fold increase upon anergy induction. Amongthose, 90 probes met the inventors' defined criteria forEgr2-dependence, in that the elevated gene expression seen in anergy wasreduced by more than 1.5-fold in Egr2-deleted cells.

To identify the direct transcriptional targets of Egr2, the inventorsalso performed a ChIP-seq analysis. CAR Tg×Egr2^(flox/flox) Th1 T cellswere untreated or anergized by immobilized anti-CD3, cross-linked, andnuclear exacts were immunoprecipated by an anti-Egr2 Ab. ChIP-seqanalysis was carried out on the DNA fragments bound to Egr2. Egr2 wasfound to be associated with a variety of regulatory regions in thegenome of anergic T cells, of which 13% were located in core promoters(50 by upstream of transcription start site (TSS)), 40% in proximalpromoters (500 by upstream of TSS), 20% in 5′UTRs, and 21% in introns(Figure. 1A). These binding interactions appeared to be specific, sincethe consensus sequence of Egr2 binding site derived from ChIP-seq washighly similar to that published on TRANSFAC, a comprehensivetranscription factor database (FIG. 1B).

When the gene array results were merged with the ChIP-seq data, 62 ofthe 90 probes that showed Egr2-dependency for their expression werefound to be directly bound by Egr2. Because of some duplication, those62 probes represented 49 genes (FIG. 1C). In summary, 2.08% (938/45101)of the gene array probes demonstrated upregulation upon anergyinduction, among which 9.59% (90/938) were dependent on Egr2, and 6.61%(62/938) were directly regulated by Egr2.

Example 3—Characterization of Novel Targets of Egr2 in the Anergic TCells

Previous mechanistic studies of T cell anergy have focused attention onidentifying the key factors that are causal for T cell intrinsicdysfunction. However, a striking feature of the additional genesidentified through the gene array/ChIP-seq analyses is that several ofthe gene products are surface molecules or secreted factors. Thisobservation suggests that anergic T cells may have additional functionalproperties oriented towards other cellular components of the immunesystem.

Nine of the newly identified Egr2-dependent targets were studied in moredetail, including the chemokine CCL1; five cell surface receptors(Semaphorin 7A (Sema7A), Class-I-MHC-restricted T cell associatedmolecule (Crtam), lymphocyte-activation gene 3 (Lag3), tumor necrosisfactor receptor superfamily member 9 (Tnfrsf9, or 4-1BB), and Neuritin(Nrn1)); and three intracellular proteins (BCL2-like 11 (Bcl2111 orBim), Neurogranin (Nrgn), and cellular retinoic acid-binding protein 2(Crapb2)). CCL1 is a member of the C—C motif chemokine family, and arecent publication indicated that CCL1 can recruit Foxp3⁺ regulatory Tcells in the tumor context (Hoelzinger et al., 2010). Semaphorin 7A(Sema7A) belongs to membrane-bound Semaphorin family that associateswith the plasma membrane via a GPI linker (Suzuki et al., 2008). Crtamis a type I transmembrane protein with V and C1-like Ig domains (DuPasquier, 2004). Lag3, a CD4-related transmembrane protein, binds to MHCclass II on APCs with higher affinity than does CD4 and has beenreported to function as an inhibitory receptor (Baixeras et al., 1992;Grosso et al., 2007; Huang et al., 2004; Triebel et al., 1990). 4-1BB isan inducible costimulation receptor on T cells, and belongs to the tumornecrosis factor receptor superfamily (Watts, 2005). Nrn1 is a neuralactivity-regulated gene, encoding a small extracellular protein thatserves as a neurotrophin to promote neuritogenesis, neuronal survival,and plasticity (Naeve et al., 1997; Nedivi et al., 1996; Nedivi et al.,1998). Nrgn is another protein that has been mainly studied in thecentral neural system (Diez-Guerra, 2010). It interacts with calmodulinand regulates intracellular concentrations of calcium, andcalcium-derived signaling in synapses. The functions of Nrn1 and Nrgn inthe immune system are yet to be clarified. Bcl2111 is a pro-apoptosisproteins in the intrinsic apoptosis pathway, and has been shown toregulate T cell deletion in both thymus and the peripheral (Bouillet andO'Reilly, 2009). Its interaction with pro-survival protein Bcl-2releases BCL-2-associated X protein (BAX) and BCL-2 antagonist/killer(BAK) to trigger mitochondria-mediated apoptosis. Crabp2 is a retinoicacid binding protein, delivering retinoid acid from the cytoplasm to itsselective receptors located in the nucleus (Hall et al., 2011).

Binding of Egr2 to theses nine genes upon anergy induction was confirmedby ChIP assay. As shown in FIG. 2, Egr2 was associated with variableregulatory regions of these genes upon immobilized anti-CD3 treatment.Egr2-dependent mRNA expression in anergic T cells was confirmed byqRT-PCR. As seen in FIG. 3, TCR engagement alone highly upregulatedthese genes, and their expressions were reduced substantially with Egr2deletion. Similar results were also seen in two other CARTg×Egr2^(flox/flox) Th1 T cell clones (FIGS. 5 and 6). To furthervalidate the qRT-PCR results, the inventors analyzed the expression offour gene products at protein level, as there were reagents availablefor this analysis. ELISA revealed that CCL1 was constitutively secretedby anergic cells in an Egr2-dependent manner (FIG. 4A). The expressionof Sema7A and Crapb2 proteins were detected by immunoblot in anergiccells, which were partially reduced in the absence of Egr2 (FIGS. 4B and4D). Similarly, cell surface expression of Crtam was detected by flowcytometry on at least 14.60±1.88% anergic cells, and diminished to3.41±0.2% when Egr2 was deleted (FIG. 4C represents one of sixexperiments). These results indicate that Egr2 directly contributes tothe expression of these nine genes in anergic cells.

Several genes encoding negative regulatory molecules are upregulated inanergic T cells (see FIGS. 7-9). Such genes include, for example, DGK-ζ,Cbl-b, GRAIL, Itch, Tob1, and Deltex1. It was also demonstrated thatEgr2-deleted T cells are relative anergy-resistant in vitro (see FIG.10) and also in vivo (data not shown). Additional genes regulated byEgr2 were identified using strategies including real-time PCR and ChIPassays, as well as gene expression profiling of conditional Egr2-deletedT cells with ChIP-SEQ analysis of genes directly binding to Egr2. Theseexperiments demonstrated that Egr2 is a major transcriptional regulatorof the anergic state and identified the Egr2 transcriptome in T cellanergy (see FIGS. 11-14). Further studies showed that Crtram(Class-I-MHC-restricted T cell associated molecule) and Lag3(lymphocyte-activation gene 3) are Egr2-dependent anergy-associatedgenes (see FIG. 15) and may identify the population of anergic T cellsfrom the tumor microenvironment ex vivo (see FIGS. 16-20).

Example 4—Characterization of Novel Targets of Egr2 in the Anergic TCells

Female C57BL/6 mice purchased from Taconic were injected with 2×10^6B16.SIY subcutaneously (Day 0). Beginning on Day 4, mice were injectedwith 100 μg of either anti-4-1BB (4-1BB antibody clone LOB12.3) oranti-LAG-3 antibody (LAG-3 antibody clone C9B7W), or 100 μg ofanti-4-1BB antibody plus 100 μg of anti-LAG-3 antibody. Mice receivedtreatments on Days 4, 7, 10 and 13. Tumors were measured beginning onDay 7. Tumor areas were calculated as the product of the longest tumordiameter and the diameter perpendicular to that. Tumors were measuredafter Day 7 on the indicated days.

4-1BB and LAG-3 single antibody treatments both provided partial tumorcontrol, however this control was temporary and tumors regrew. Combinedtreatment with 4-1BB and LAG-3 antibodies provided very robust tumorcontrol, with 60% of mice in this group having no palpable tumor 30 daysafter tumor injection (FIG. 21). This demonstrates that combiningtreatments against two or more anergy associated targets can markedlyimprove immune-mediated tumor control.

TABLE 1 Average Upreg- Upreg- Upreg- Upreg- ulation ulation ulationulation Accession Entrez Gene Control 1 Anergic 1 1 (fold) Control 2Anergic 2 2 (fold) Control 3 Anergic 3 3 (fold) (fold) NM_019465 5469837.62 6042.76 160.62 41.68 2843.67 68.23 36.94 2524.99 68.36 99.07AF128196 20308 16.45 1080.96 65.73 16.11 376.95 23.4 28.27 2210.41 78.1955.77 AK003046 68404 198.49 6839.14 34.46 119.04 8426.23 70.78 178.634604.82 25.78 43.67 NM_011329 20290 100.28 6885.64 68.67 132.65 2909.6921.93 86.35 1939.45 22.46 37.69 X06746 13654 119.94 4756.51 39.66 71.982490.8 34.6 82.73 2183.82 26.4 33.55 X06746 13654 163.45 6015.28 36.8114.43 3489.41 30.49 114.33 3120.32 27.29 31.53 NM_009375 21819 71.772168.62 30.22 114.04 1949.49 17.1 98.42 3311.95 33.65 26.99 NM_01037214941 44.37 660.44 14.89 138.37 3081.58 22.27 29.81 1010.2 33.89 23.68U94828 19734 35.03 616.21 17.59 63.44 2178.53 34.34 37.77 424.3 11.2321.05 NM_010372 14941 49.16 716.56 14.57 169.47 3336.17 19.69 38.061092.99 28.72 20.99 NM_009877 12578 17.32 451.49 26.07 47.03 1223.1126.01 27.67 300.22 10.85 20.98 AF128196 20308 95.12 1597.89 16.8 97.88650.4 6.64 102.47 3631.53 35.44 19.63 NM_013542 14939 238.24 2842.2611.93 185.57 1756.16 9.46 249.25 7554.5 30.31 17.23 M12573 15511 58.181154.01 19.83 25.23 446.53 17.7 69.25 941.26 13.59 17.04 NM_011337 20302267.54 2340.01 8.75 95.01 2970.04 31.26 436.58 3000.83 6.87 15.63 U7288119734 43.4 448.32 10.33 47.31 1311.74 27.73 41.48 340.96 8.22 15.43NM_011454 20708 95.92 1908.44 19.9 66.89 1117.49 16.71 114.43 962.328.41 15.01 M12573 15511 104.73 1706.78 16.3 44.57 639.61 14.35 108.021439.44 13.33 14.66 AV309418 17988 126.65 588.61 4.65 103.03 311.64 3.0282.66 2859.45 34.59 14.09 M13227 18619 1006.56 12533.48 12.45 819.6211806.54 14.4 917.86 11977.63 13.05 13.30 AI987929 17988 87.36 379.514.34 52.76 226.81 4.3 63.48 1953.73 30.78 13.14 NM_138648 108078 13.37208.34 15.59 17.29 351.86 20.35 30.73 90.09 2.93 12.96 AV162459 68612415.52 2065.66 4.97 60.81 1392.81 22.9 255.58 1774.24 6.94 11.60BC015076 14012 40.73 313.46 7.7 34.11 296.98 8.71 24.3 429.15 17.6611.36 AK013312 12442 490.1 1992.99 4.07 63.95 1495.86 23.39 269.591774.29 6.58 11.35 BC004702 11799 400.81 2084.32 5.2 89.3 1616.32 18.1206.75 2157.13 10.43 11.24 M12573 15511 173.32 2243.94 12.95 87.83778.33 8.86 170.01 1903.83 11.2 11.00 NM_013653 20304 583.38 5365.47 9.2395.69 6362.05 16.08 795.22 5396.55 6.79 10.69 NM_010212 14200 73.631229.38 16.7 126.57 997.21 7.88 85.75 626.59 7.31 10.63 AK009012 71884160.41 2180.34 13.59 169.77 1960.02 11.54 143.48 957.14 6.67 10.60BC010581 100039888 /// 285.63 1341.21 4.7 45.51 783.34 17.21 142.881382.81 9.68 10.53 16765 /// 623112 AV204216 242341 18.63 137.93 7.425.5 403.36 15.82 34.54 284.71 8.24 10.49 BC015076 14012 36.83 251.326.82 32.7 254.87 7.79 20.29 339.99 16.76 10.46 NM_009129 20254 15.16241.74 15.94 15.53 157.86 10.16 16.49 85.08 5.16 10.42 BC028507 21942136 958.64 7.05 149.19 499.87 3.35 139.55 2898.97 20.77 10.39 NM_02647367951 240.2 1791.56 7.46 308.51 1937.32 6.28 207.67 3562.59 17.15 10.30AK017673 68026 703.37 3223.15 4.58 153.15 2589.14 16.91 421.28 3744.578.89 10.13 AK009873 76459 58.98 903.18 15.31 54.53 250.94 4.6 58.27607.31 10.42 10.11 NM_025581 66468 52.99 268.85 5.07 12.21 206.91 16.9533.86 278.11 8.21 10.08 BC003738 19362 72.54 382.74 5.28 24.04 252.9110.52 32.7 463.8 14.18 9.99 AK002933 64011 384.24 4596.58 11.96 333.634493.08 13.47 432.07 1930.41 4.47 9.97 AI987929 17988 111.9 463.02 4.14106.91 298.43 2.79 100.56 2305.13 22.92 9.95 AV294537 239650 263.372861.61 10.87 190.96 1518.64 7.95 334.61 3552.8 10.62 9.81 C77256 110611186.22 1686.15 9.05 166.14 1582.19 9.52 183.92 1919.75 10.44 9.67BE985366 77619 90.12 560.93 6.22 67.79 424.73 6.26 96.79 1495.3 15.459.31 BM120925 12125 42.65 701.34 16.45 47.52 338.77 7.13 49.6 203.624.11 9.23 AK017673 68026 105.53 598.72 5.67 39.38 389.28 9.89 61.16731.44 11.96 9.17 X75483 12428 364.43 1549.19 4.25 69.21 1015.95 14.68204.84 1654.58 8.08 9.00 NM_033597 17863 36.94 216.46 5.86 44.76 358.838.02 27.94 345.1 12.35 8.74 NM_011613 21943 109.59 1750.01 15.97 275.911776.93 6.44 101.84 381.19 3.74 8.72 NM_011617 21948 229.61 1813.34 7.9107.78 1024.93 9.51 173.04 1504.58 8.69 8.70 NM_010790 17279 88.81558.66 6.29 26.15 292.55 11.19 63.72 532.53 8.36 8.61 BC009096 108907338.25 1625.69 4.81 83.68 1048.33 12.53 217.18 1795.07 8.27 8.54BC010581 16765 1589.94 5253.41 3.3 285.86 4584.76 16.04 844.7 5234.966.2 8.51 BE981853 72309 105.46 1198.18 11.36 78.48 555.6 7.08 166.51180.27 7.09 8.51 NM_026358 67749 52.99 244.07 4.61 93.3 110.76 1.1954.62 1053.72 19.29 8.36 NM_011799 23834 56.89 308.38 5.42 26.33 211.148.02 34.52 399.71 11.58 8.34 X75483 12428 395.8 1925.18 4.86 103.471234.27 11.93 284.52 2247.04 7.9 8.23 NM_134066 105349 105.79 513.484.85 93.6 598.67 6.4 119.9 1593.1 13.29 8.18 AK010391 71988 59.77 253.544.24 11.43 133.49 11.68 35.39 294.36 8.32 8.08 BG065877 110611 404.342371.57 5.87 218.1 1899.09 8.71 371.62 3582.73 9.64 8.07 AK009549 69621243.71 1457.61 5.98 114.55 1103.83 9.64 231.17 1962.67 8.49 8.04BB356493 654795 107.58 846.26 7.87 160.05 1622.31 10.14 117.17 667.055.69 7.90 NM_008479 16768 145.86 1046.69 7.18 138.03 1210.26 8.77 232.451652.41 7.11 7.69 BE979441 330662 259.22 1501.19 5.79 180.93 1288.547.12 231.44 2247.66 9.71 7.54 NM_008681 17988 193 691.02 3.58 202.57552.85 2.73 194.3 3129.69 16.11 7.47 D87867 22236 /// 103.38 637.31 6.1780.92 987.9 12.21 114.37 413.07 3.61 7.33 394430 /// 394432 /// 394433/// 394434 /// 394435 /// 394436 /// 94284 AK011162 72107 21.57 129.32 611.23 83.08 7.4 16.98 140.55 8.28 7.23 AV332575 70564 141.07 1117.277.92 120.96 843.29 6.97 115.35 756.49 6.56 7.15 NM_009104 20135 261.791316.88 5.03 84.37 546.76 6.48 159.92 1531.79 9.58 7.03 BB702047 52276248.32 1058.73 4.26 64.89 680.43 10.49 154.01 953.59 6.19 6.98 BC019755213696 266.1 2191.29 8.23 308.32 1938.47 6.29 192.91 1218.64 6.32 6.95AU015121 268697 311.47 1320.63 4.24 62.93 668.42 10.62 211.69 1211.515.72 6.86 BC003738 19362 32.48 197.24 6.07 16.89 126.7 7.5 32.22 219.856.82 6.80 BB667581 12125 101.08 1212.68 12 128.43 592.15 4.61 102.45350.73 3.42 6.68 NM_080853 140919 547.69 2798.74 5.11 103.84 984.82 9.48386.53 2088.4 5.4 6.66 AK006467 75568 38.97 234.43 6.02 38.7 239.93 6.233.68 251.2 7.46 6.56 NM_009127 20249 83.01 307.56 3.7 84.01 539.87 6.4350.59 481.41 9.52 6.55 NM_010555 16178 220.2 1421.28 6.45 660.3 2809.924.26 213.37 1889.76 8.86 6.52 NM_011369 20419 121.86 709.24 5.82 58.76307.36 5.23 87.15 736.33 8.45 6.50 NM_007669 12575 654.48 3378.76 5.16697.59 2253.54 3.23 393.85 4366.21 11.09 6.49 NM_010373 14942 131.1520.52 3.97 254.26 2076.29 8.17 109.3 790.59 7.23 6.46 NM_009104 20135326.79 1684.91 5.16 115.63 720.92 6.23 230.46 1806.55 7.84 6.41 BQ180367140919 668.86 4182.56 6.25 199.3 1525.01 7.65 562.26 2955.6 5.26 6.39NM_019631 56277 97.93 434.11 4.43 49.54 96.37 1.95 73.28 936.83 12.786.39 AV301324 20135 643.82 2928.78 4.55 193.86 1168.64 6.03 350.973006.49 8.57 6.38 AK010351 66977 134.31 633.64 4.72 36.39 249.61 6.8685.79 649.26 7.57 6.38 BC018397 12904 79.73 473.88 5.94 65.82 303.954.62 71.5 611.36 8.55 6.37 AV251613 97086 33.44 234.53 7.01 39.8 221.315.56 39.1 252.86 6.47 6.35 AF450241 171285 742.48 2936.35 3.95 162.981564.93 9.6 514.93 2780.8 5.4 6.32 BC027121 66442 202.22 893.22 4.4271.03 470.33 6.62 113.65 889.74 7.83 6.29 NM_007659 12534 523.86 1793.763.42 122.12 1093.38 8.95 301.74 1938.8 6.43 6.27 NM_007629 12429 ///164.48 661.2 4.02 33.14 315.55 9.52 111.41 566.16 5.08 6.21 268697 ///434175 /// 667005 AB032771 21943 103.06 956.93 9.29 149.31 899 6.0279.46 262.5 3.3 6.20 AY083458 235505 115.89 656.57 5.67 35.14 216.086.15 139.05 936.58 6.74 6.19 NM_009127 20249 153.48 563.53 3.67 134.68772.61 5.74 121.44 1085.82 8.94 6.12 AF032460 12125 44.82 346.31 7.7331.54 208.29 6.6 33.85 134.56 3.98 6.10 BI081061 14793 285.63 1212.944.25 135.14 1064.1 7.87 209.58 1275.22 6.08 6.07 AK018120 70727 22.14235.21 10.63 16.39 77.13 4.71 27.56 73.73 2.68 6.01 NM_011234 19361187.22 822.96 4.4 71.59 380.27 5.31 119.49 992.27 8.3 6.00 NM_02320952033 149.65 629.07 4.2 46.98 293.25 6.24 77.43 584.91 7.55 6.00BB375974 80752 24.28 133.21 5.49 21.54 222.77 10.34 36.27 78 2.15 5.99AK014919 66336 51.39 298.93 5.82 36.02 259.75 7.21 57.28 280.34 4.895.97 BB702754 18140 152.44 635.09 4.17 51.85 418.46 8.07 135.16 763.425.65 5.96 BC019946 11910 135.01 551.19 4.08 221.82 1560.81 7.04 127.55861.08 6.75 5.96 NM_010558 16191 293.56 907.63 3.09 62.96 99.57 1.58178.49 2302.56 12.9 5.86 NM_009004 19348 69.7 387.85 5.56 27.71 144.255.21 54.25 367.05 6.77 5.85 BB758432 330938 58.12 238.71 4.11 22.1 61.32.77 72.05 756.32 10.5 5.79 AF079222 15366 64.93 343.18 5.29 36.41159.42 4.38 51.83 398.54 7.69 5.79 BC005799 76131 52.48 331.47 6.3220.56 134.17 6.53 52.15 221.64 4.25 5.70 AI266795 20193 129.19 769.65.96 167.03 772.77 4.63 135.34 844.69 6.24 5.61 NM_010373 14942 154.2501.86 3.25 280.61 1951.09 6.95 114.74 759.38 6.62 5.61 AF002823 12235127.53 521.08 4.09 53.1 277.79 5.23 93.8 703.2 7.5 5.61 AK017688 70564357.22 2074.29 5.81 299.33 1658.12 5.54 326.1 1769.95 5.43 5.59 AA98608273710 55.28 224.04 4.05 84.36 216.47 2.57 36.42 366.78 10.07 5.56AF181829 56193 169.67 663.63 3.91 78.35 691.32 8.82 170.92 674.32 3.955.56 AF181829 56193 99.78 436.09 4.37 51.65 411.88 7.97 100.38 435.714.34 5.56 AV132173 12615 382.48 1705.09 4.46 160.76 1346.28 8.37 314.951198.37 3.8 5.54 BC005773 26558 61.33 216.63 3.53 47.82 204.31 4.2744.22 389.48 8.81 5.54 NM_011182 19159 64.08 729.63 11.39 200.3 543.552.71 68.77 171.94 2.5 5.53 AK007630 12575 576.19 3112.8 5.4 852.892146.96 2.52 487.14 4120.42 8.46 5.46 NM_016900 12390 275.34 1377.31 5218.04 1028.07 4.71 247.57 1646.46 6.65 5.45 NM_009647 100047616 ///111.03 244.55 2.2 79.07 92.72 1.17 95.85 1236.26 12.9 5.42 11639BC003261 20877 134.54 578.16 4.3 57.3 327.17 5.71 128.34 781.35 6.095.37 NM_007629 12429 /// 233.48 863.03 3.7 86.74 632.05 7.29 151.2754.06 4.99 5.33 268697 AF079222 15366 51.38 317.21 6.17 19.68 94.134.78 50.98 254.78 5 5.32 C77054 18005 148.22 566.54 3.82 59.48 432.437.27 104.8 503.06 4.8 5.30 NM_007691 12649 34.73 209.75 6.04 27.09 95.353.52 32.09 203.18 6.33 5.30 BI794748 230259 86.29 532.49 6.17 167.4727.36 4.34 112.41 601.24 5.35 5.29 BB703394 47.36 185.52 3.92 70.98593.83 8.37 57.74 203.46 3.52 5.27 AK010477 69745 658.21 3244.24 4.93415.69 2540.46 6.11 568.21 2699.31 4.75 5.26 NM_009707 11856 22.39124.25 5.55 10.59 43.27 4.09 22.53 137.43 6.1 5.25 NM_009760 12176833.25 2306.52 2.77 587.18 886.04 1.51 600.54 6848.94 11.4 5.23NM_012057 27056 160.55 1072.57 6.68 109.64 369.9 3.37 233.05 1300.135.58 5.21 BG060909 20250 428.25 1305.05 3.05 460.04 1107.78 2.41 358.993643.45 10.15 5.20 NM_033597 17863 58.16 269.49 4.63 82.37 419.35 5.0975 429.94 5.73 5.15 BC003261 20877 126.58 459.97 3.63 39.52 230.56 5.8396.12 555.56 5.78 5.08 NM_009741 100046608 /// 149.06 922.54 6.19 218.84973.19 4.45 184.66 845.05 4.58 5.07 12043 NM_133888 100340 269.5 1473.265.47 214.19 1171.54 5.47 278.26 1174.95 4.22 5.05 NM_016851 54139 73.8326.02 4.42 60.6 413.6 6.83 49.4 188.74 3.82 5.02 NM_009014 19363 61.66287.21 4.66 53.04 285.79 5.39 50.17 251.05 5 5.02 BB041150 107995 467.251746.63 3.74 138.32 920.52 6.65 292.06 1330.31 4.55 4.98 BB463610 7404164.78 280.92 4.34 61.27 187.95 3.07 40.61 298.88 7.36 4.92 AF03296927.64 160.53 5.81 35.35 162.81 4.61 31.07 133.91 4.31 4.91 AV084342654824 193.07 507.23 2.63 74.93 106.64 1.42 174.24 1854.31 10.64 4.90AV224521 227753 504.64 1292.45 2.56 397.19 985.5 2.48 382.53 3640.689.52 4.85 BM250782 21942 354.82 1548.21 4.36 418.65 1032.68 2.47 470.13625.74 7.71 4.85 AA709993 20365 89.93 345.76 3.84 58.77 355.27 6.0470.99 327.4 4.61 4.83 BB408123 66214 44.38 158.78 3.58 78.2 345.34 4.4240.69 262.38 6.45 4.82 BI731319 23969 44.37 346.19 7.8 51.5 246.35 4.7851.3 92.14 1.8 4.79 BC019416 94346 104.43 475.81 4.56 95.46 434.58 4.55102.19 531.11 5.2 4.77 X64550 15366 83.03 442.5 5.33 48.14 181.94 3.7878.67 408.55 5.19 4.77 BG060909 20250 135.92 395.57 2.91 111.62 288.742.59 114.14 997.74 8.74 4.75 BB497312 114644 85.76 279.09 3.25 73.61326.75 4.44 85.39 556.34 6.52 4.74 AK010166 71934 39.79 180.41 4.53 33.6200.49 5.97 45.28 166.57 3.68 4.73 BB827235 16551 102.37 428.5 4.1958.45 244.1 4.18 69.27 399.73 5.77 4.71 BB100249 19734 290.68 1034.963.56 312.77 2444.97 7.82 287.14 769.47 2.68 4.69 AV307110 52276 243.71839.6 3.45 97.67 563.34 5.77 178.02 856.73 4.81 4.68 BM120925 12125100.34 893.96 8.91 109.98 321.17 2.92 121.24 261.75 2.16 4.66 AF00402317470 540.63 2623.9 4.85 364.26 1805.23 4.96 494.9 2037.46 4.12 4.64BC016135 14697 213.79 1156.12 5.41 172.76 777.31 4.5 176.91 711.47 4.024.64 AA197362 70466 177.07 727.74 4.11 84.22 392 4.65 136.2 700.24 5.144.63 AW763765 193740 45.76 227.35 4.97 37.81 136.79 3.62 48.26 254.385.27 4.62 NM_016904 54124 410.15 1540.06 3.75 239.83 1161.3 4.84 271.691413.88 5.2 4.60 BM234447 16551 153.6 574.81 3.74 56.65 258.16 4.56113.73 605.07 5.32 4.54 BC006674 70385 103.38 437.72 4.23 60.65 204.883.38 78.23 470.2 6.01 4.54 NM_025569 66447 295.5 969.39 3.28 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155.16 1.75128.1 417.77 3.26 84.37 132.52 1.57 2.19 NM_026410 67849 441.78 958.422.17 356.73 717.13 2.01 464.14 1107.49 2.39 2.19 BG965431 27388 272.57499.47 1.83 170.36 478.96 2.81 284.24 549.97 1.93 2.19 AF059029 108058121.47 262.01 2.16 137.67 304.38 2.21 124.7 273.71 2.19 2.19 BB20091160.69 87.55 1.44 108.51 295.29 2.72 71.84 171.74 2.39 2.18 BM20758820525 870.24 1561.19 1.79 1316.83 1212.54 −1.09 785.5 4594.38 5.85 2.18BB095626 67458 397.22 849.24 2.14 354.66 724.22 2.04 313.98 745.38 2.372.18 BG066131 233806 350.16 878.11 2.51 352.39 776.93 2.2 340.17 627.151.84 2.18 NM_010371 14940 122.87 207.98 1.69 86.43 168.74 1.95 114.57331.58 2.89 2.18 BB133021 320209 116.24 267.26 2.3 71.57 164.72 2.3100.56 194.1 1.93 2.18 NM_023707 100040208 /// 63.8 144 2.26 111.99252.52 2.25 75.38 152.61 2.02 2.18 100044143 /// 73626 NM_020282 18105863.39 1299.75 1.51 225.82 735.58 3.26 934.45 1634.43 1.75 2.17 BB62193899382 320.32 632.51 1.97 531.76 1294.36 2.43 325.09 690.56 2.12 2.17AI837704 234593 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2.15NM_019780 56433 1339.3 2520.41 1.88 812.6 2249.34 2.77 1374.47 2478.481.8 2.15 NM_030254 80286 607.71 1556.78 2.56 634.28 1496.83 2.36 736.271129.41 1.53 2.15 BE692107 18124 128.53 330.88 2.57 91 242.3 2.66 131.45159.47 1.21 2.15 NM_018734 55932 576.2 1177.43 2.04 380.97 1041.87 2.73943.14 1575.89 1.67 2.15 AK002362 17918 498.15 1013.69 2.03 425.851035.34 2.43 450.7 893.23 1.98 2.15 BB268139 268291 292.17 478.62 1.64198.75 316.09 1.59 241.15 773.11 3.21 2.15 BM118729 75007 134.38 310.852.31 112.38 245.67 2.19 168.89 327.13 1.94 2.15 BC008166 100039683 ///541.8 1014.2 1.87 352.22 960.16 2.73 478.75 875.54 1.83 2.14 100045937/// 100047518 /// 76281 AK003874 71793 252.16 556.25 2.21 337.94 632.591.87 254.23 597.44 2.35 2.14 BI658203 212999 158.79 433.18 2.73 128.53228.94 1.78 164.14 315.65 1.92 2.14 BG070404 76843 60.74 151.96 2.573.45 141.32 1.92 63.15 127.06 2.01 2.14 NM_019999 56695 448.99 728.931.62 217.42 674.88 3.1 480.31 818.21 1.7 2.14 BC010332 50784 345.58608.07 1.76 291.95 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NM_009929 12822 93.5 167.48 1.79 119.46 139.151.16 95.94 319.36 3.33 2.09 BB458835 30926 1904.97 3577.2 1.88 1423.813262.87 2.29 1855.3 3888.38 2.1 2.09 AA590970 29816 417.25 871.49 2.09404.66 1077.33 2.66 648.07 987.57 1.52 2.09 NM_011132 18973 157.76303.81 1.93 130.9 239.58 1.83 136.81 342.77 2.51 2.09 BC016456 69459500.33 1318.78 2.64 398.24 991.56 2.49 810.56 918.35 1.13 2.09 AF22943421936 3829.32 6414.36 1.68 2319.14 5236.34 2.26 3630.77 8432.08 2.322.09 AV020390 22333 1586.75 2743.35 1.73 1440.29 2343.62 1.63 1492.224334.1 2.9 2.09 BC003432 110842 1260.28 2260.06 1.79 719.58 1915.71 2.661214.77 2196.63 1.81 2.09 BG064103 22029 1657.49 2001.82 1.21 276.651053.75 3.81 1696.66 2103.28 1.24 2.09 BE197524 14469 921.3 1466.75 1.59427.68 1264.7 2.96 1020.19 1739.73 1.71 2.09 NM_007657 12527 258.3640.17 2.48 743.51 1811.34 2.44 281.15 375.66 1.34 2.09 NM_021477 268859216 435.03 2.01 66.74 175.65 2.63 196.48 318.55 1.62 2.09 NM_01674120778 154.46 300.25 1.94 123.02 280.83 2.28 164.44 334.92 2.04 2.09BM234652 50527 1284.17 1549.71 1.21 544.42 717.48 1.32 1200.86 4463.113.72 2.08 BI151440 12495 510.71 615.15 1.2 222.7 240.05 1.08 362.981442.58 3.97 2.08 AV302436 665401 /// 224.87 495.37 2.2 192.1 420.472.19 193.91 361.17 1.86 2.08 67383 /// 677553 BC009150 67286 172.24281.69 1.64 137.49 297.34 2.16 133.09 326.15 2.45 2.08 AK003718 15258538.29 1402.81 2.61 413.83 1003.86 2.43 702.68 843.39 1.2 2.08 BC017138109168 182.03 337.79 1.86 158.53 344.31 2.17 179.27 396.94 2.21 2.08NM_021782 547223 /// 138.6 127.85 −1.08 64.67 322.94 4.99 173.12 402.732.33 2.08 60505 /// 637754 BB093469 69941 103.7 203.05 1.96 110.93170.58 1.54 104.73 286.97 2.74 2.08 NM_009895 12700 769.56 2010.58 2.612197.33 3217.86 1.46 816.94 1764.37 2.16 2.08 AV280756 207607 186.17354.22 1.9 130.26 323.17 2.48 186.86 344.84 1.85 2.08 AW553304 1201469.22 130.87 1.89 108.51 592.51 5.46 74.69 66.87 −1.12 2.08 AU01988089.44 275.12 3.08 79.87 120.44 1.51 139.26 228.92 1.64 2.08 BB093351381306 60.16 106.61 1.77 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2.06 2.07 NM_01999056018 301.65 748.92 2.48 498.47 706.01 1.42 381.8 876.16 2.29 2.06BB458178 105501 1619.06 2764.03 1.71 838.26 2302.2 2.75 1393.3 2392.591.72 2.06 AV059518 67117 1004.35 2096.53 2.09 812.5 1495.08 1.84 875.541966.67 2.25 2.06 NM_016761 100042970 /// 150.11 263.77 1.76 116.64202.4 1.74 114.01 305.41 2.68 2.06 100044006 /// 100044746 /// 16580BB364961 94.25 185.35 1.97 126.27 227.47 1.8 104.46 250.5 2.4 2.06BF303544 56491 303.74 827.85 2.73 579.36 1186.27 2.05 714.05 981.92 1.382.05 BC008238 56791 64.44 149.3 2.32 74.24 167.46 2.26 62.27 98.28 1.582.05 BB449218 67071 47.01 87.24 1.86 72.29 149.22 2.06 52.92 118.36 2.242.05 AK010005 71924 97.02 200.95 2.07 77 124.61 1.62 107.26 263.9 2.462.05 BE988432 65247 81.76 181.22 2.22 94.4 179.31 1.9 100.41 203.97 2.032.05 AV328388 17196 80.37 183.33 2.28 165.49 179.66 1.09 60.5 168.212.78 2.05 BF466605 66337 86.7 179.45 2.07 75.43 176.79 2.34 90.6 157.561.74 2.05 AF237702 20425 78.83 161.9 2.05 95.54 142.64 1.49 73.59 192.352.61 2.05 NM_011303 20148 441.15 1194.82 2.71 484.38 850.3 1.76 658.591105.49 1.68 2.05 NM_007522 12015 219.43 377.29 1.72 121.38 294.96 2.43193.24 384.13 1.99 2.05 BE950435 74023 83.79 176.86 2.11 91.21 138.741.52 81.33 204.17 2.51 2.05 NM_010585 16438 886.88 1569.08 1.77 555.31047.89 1.89 786.94 1946.85 2.47 2.04 BI658203 212999 246.06 666.61 2.71201.82 296.34 1.47 309.75 603.72 1.95 2.04 BB283759 12995 169.17 320.391.89 149.9 272.91 1.82 158.89 383.65 2.41 2.04 NM_024223 68337 134.34302.57 2.25 168.69 230.75 1.37 124.29 310.88 2.5 2.04 AI461712 70984186.98 397.5 2.13 106.63 153.04 1.44 146.56 373.65 2.55 2.04 BC00816168533 837.11 1573.77 1.88 1771.7 2217.11 1.25 761.56 2269.65 2.98 2.04NM_021451 58801 768.72 1937.88 2.52 1358.05 1745.57 1.29 959.46 2206.592.3 2.04 X58876 17246 852.2 1607.24 1.89 779.75 1578.04 2.02 839.51850.15 2.2 2.04 AK015214 67138 386.78 642.92 1.66 218.2 565.61 2.59364.51 678.97 1.86 2.04 BC028439 66395 283.56 606.1 2.14 401.36 617.711.54 270.17 655.51 2.43 2.04 AU019491 51944 338.52 699.66 2.07 269.43534.46 1.98 309.96 637.11 2.06 2.04 BC006736 75430 205.52 392.31 1.91226.36 366.34 1.62 175.15 452.11 2.58 2.04 BE952632 26378 215.19 422.631.96 140.13 294.26 2.1 179.78 368.52 2.05 2.04 BB751158 17454 152.38286.13 1.88 181.48 269.77 1.49 192.06 526.42 2.74 2.04 BB782705 14679116.44 380.3 3.27 224.41 391.87 1.75 197.19 215.7 1.09 2.04 NM_00998013017 154.06 318.08 2.06 79.52 176.96 2.23 187.47 340.63 1.82 2.04AF196480 23947 126.41 259.97 2.06 83.81 186.31 2.22 122.17 222.16 1.822.03 BQ031123 66264 102.05 232.32 2.28 112.64 221.97 1.97 95.48 176.311.85 2.03 BB770972 100047967 /// 87.95 162.77 1.85 75.82 132.03 1.7469.54 174.47 2.51 2.03 237436 BB114398 12816 85.23 147.26 1.73 64.54166.23 2.58 75.77 135.04 1.78 2.03 BB315555 18537 1525.52 2402.91 1.58615.47 1821.05 2.96 1365.82 2119.74 1.55 2.03 NM_024194 67144 319.91663.11 2.07 281.38 456.04 1.62 271.37 650 2.4 2.03 BB706079 20873 185.78401.68 2.16 145.07 281.29 1.94 176.97 351.8 1.99 2.03 AK006658 73327146.58 352.15 2.4 157.48 265.95 1.69 151.13 301.73 2 2.03 BC015270 9711459.74 120.94 2.02 40.71 61.97 1.52 59.26 151.41 2.55 2.03 NM_00783013167 1090.59 1828.06 1.68 798.3 1502.71 1.88 861.53 2174.24 2.52 2.03BC019420 70544 575.76 1219.78 2.12 586.76 1169.57 1.99 527.67 1039.221.97 2.03 BB039269 14609 144.89 264.86 1.83 85.6 182.53 2.13 103.11218.96 2.12 2.03 BB120594 30957 90.31 174.93 1.94 60.48 104.55 1.7377.59 187.1 2.41 2.03 AK016473 100037282 /// 1409.46 2640.12 1.87 793.482060.87 2.6 1379.82 2212.52 1.6 2.02 66832 NM_012010 100039419 ///874.78 1524.28 1.74 652.74 1250.4 1.92 594.62 1430.83 2.41 2.02100048746 /// 26905 BG060677 223255 415.2 873.33 2.1 729.75 1318.27 1.81356.3 767.84 2.16 2.02 BB376573 68127 270.52 502.5 1.86 151.09 401.92.66 177.29 274.31 1.55 2.02 NM_019670 56419 179.53 318.58 1.77 92.77218.63 2.36 155.76 302.74 1.94 2.02 AK005139 68262 756.51 925.39 1.22396.96 749.3 1.89 579.83 1709.58 2.95 2.02 Z25469 19128 347.58 694.16 2170.15 501.31 2.95 337.85 374.69 1.11 2.02 NM_016923 17087 359.9 621.341.73 140.36 436.16 3.11 329.85 403.68 1.22 2.02 BB397062 69863 217.84456.79 2.1 277.01 420.78 1.52 161.15 390.83 2.43 2.02 BE630073 163.8309.05 1.89 101.53 263.76 2.6 193.18 302.11 1.56 2.02 AI426175 100900626.1 1256.56 2.01 569.34 1239.36 2.18 573.67 1064.89 1.86 2.02 BG06725172085 147.01 281.62 1.92 71.23 110.09 1.55 138.45 357.5 2.58 2.02NM_013832 19415 113.33 271.27 2.39 102.42 225.54 2.2 116.54 169.65 1.462.02 AK008567 67899 927.84 2002.88 2.16 1040.81 2496.92 2.4 1155.561711.06 1.48 2.01 NM_025360 66111 1018.21 2009.44 1.97 770.14 2006.652.61 1368.97 2004.86 1.46 2.01 BG063199 103080 202.82 365.1 1.8 164.91244.77 1.48 154.9 426.81 2.76 2.01 BC019957 67102 148.33 282.24 1.9139.15 270.12 1.94 150.57 331.04 2.2 2.01 BI143942 20322 330.53 768.542.33 296.29 528.36 1.78 432.9 833.21 1.92 2.01 NM_026125 67389 429.31771.39 1.8 522.64 997.74 1.91 485.65 1125.31 2.32 2.01 BB172698 69367583.96 1019.4 1.75 334.44 796.27 2.38 488.29 926.77 1.9 2.01 AK00503271779 145.08 314.45 2.17 134.29 262.74 1.96 155.49 295.59 1.9 2.01NM_133348 70025 4384.05 7588.24 1.73 2480.38 6749.69 2.72 4535.387109.42 1.57 2.01 BB324206 218236 374.74 702.68 1.88 301.06 683.13 2.27400.58 747.82 1.87 2.01 BG071041 212377 232.05 498.76 2.15 656.871044.35 1.59 216.8 494.78 2.28 2.01 BQ179335 24056 37.76 90.77 2.4 76.8790.46 1.18 49.3 120.29 2.44 2.01 BE200196 76273 2542.42 5294.28 2.082546.38 4782.64 1.88 2200.03 4520.38 2.05 2.00 NM_008229 15182 633.881065.85 1.68 390.97 1141.26 2.92 879.36 1239.53 1.41 2.00 NM_00820615001 196.35 475.03 2.42 274.64 466.48 1.7 200.48 378.75 1.89 2.00NM_019686 56506 101.08 250.03 2.47 170.56 388.02 2.27 106.61 135.09 1.272.00 AK020053 77875 94.12 118.61 1.26 94.44 130.59 1.38 97.26 327.713.37 2.00 AV355474 97.17 126.6 1.3 102.46 167.2 1.63 70.57 217.16 3.082.00 NM_008828 18655 /// 6457.37 11634.83 1.8 5061.11 8114 1.6 5753.5714971.56 2.6 2.00 668435 NM_030717 80907 608.06 1009.11 1.66 186.63556.61 2.98 550.13 747.46 1.36 2.00 BM876680 77048 324.56 735.36 2.27275.94 583.24 2.11 363.74 588.38 1.62 2.00 NM_016850 54123 279.37 335.241.2 129.76 383.74 2.96 261.54 481.08 1.84 2.00 BC010318 74551 204.82373.34 1.82 132.2 331.05 2.5 224.79 378.12 1.68 2.00

TABLE 2 Gene name Description Alternate names 1 Ccl1 chemokine (C-Cmotif) ligand 1 SCYA1, I-309, TCA3, P500, SISe 2 Crtam cytotoxic andregulatory T cell CD355 molecule 3 Egr2 early growth response 2 AT591;CMT1D; CMT4E; KROX20 4 Rasgef1a RasGEF domain family, CG48531 2 member1A FLJ378171 CG4853 gene product 5 Car12 carbonic anhydrase XII CA12carbonic anhydrase XII CAXII HsT188161 carbonic anhydrase 12 Carbonatedehydratase XII carbonic dehydratase Tumor antigen HOM-RCC- 3.1.32 3 EC4.2.1.13 CA-XII2 3 6 Fhl2 four and a half LIM domains 2 AAG11; DRAL;FHL-2; SLIM- 3; SLIM3 7 Pscd3 cytohesin 3 Cyth-3, cytohesin 31 2 ARFnucleotide-binding site opener 32 3 ARNO31 2 3 5 PH, SEC7 andcoiled-coil domain- containing protein 32 3 GRP11 2 3 5 cytohesin- 32PSCD31 2 3 5 Grp13 pleckstrin homology, Sec7 and coiled-coil domains 312 Protein ARNO33 General receptor of phosphoinositides 12 3 8 Pacsin1protein kinase C and casein protein kinase C and casein kinase substratein neurons 1 kinase substrate in neurons SDPI KIAA13793 protein kinase Cand casein kinase substrate in neurons protein syndapin I 9 Tnfrs9/4-1BBtumor necrosis factor (ligand) CD137 superfamily, member 9 10 Bcl2l11BCL2-like 11 (apoptosis BCL2-like 11 (apoptosis facilitator)facilitator) BIM-beta7 BIM1 bcl-2 interacting mediator of cell deathBOD1 bcl-2 interacting protein Bim BimEL1 bcl-2-like protein 11 BimL1bcl-2-related ovarian death agonist BimS1 bcl2-L-11 BAM2 B +C18cl2-L-113 BIM-alpha6 Bcl2-interacting mediator of cell deathBIM-beta6 11 Gnb5 guanine nucleotide binding guanine nucleotide bindingprotein (G protein), beta 5 protein (G protein), beta 5 gbeta5 GB5guanine nucleotide-binding protein subunit beta-5 Transducin beta chain5 guanine nucleotide-binding protein, beta subunit 5L G protein, betasubunit 5L Gbeta5 G protein, beta-5 subunit 12 Sdr39u1 short chain shortchain dehydrogenase/reductase dehydrogenase/reductase family 39U, member1 family 39U, member 1 C14orf124 HCDI Short-chaindehydrogenase/reductase family 39U member chromosome 14 open readingframe 124 epimerase family protein SDR39U 13 Arc activity-regulatedcytoskeleton-associated protein 14 Tnfsf11 tumor necrosis factor(ligand) tumor necrosis factor (ligand) superfamily, member 11superfamily, member 11 + D19 Receptor activator of nuclear factorkappa-B ligand OPGL TNF-related activation- induced cytokine TRANCEOPTB22 ODF hRANKL2 RANKL receptor activator of nuclear factor kappa Bligand CD254 sOdf Osteoclast differentiation factor tumor necrosisfactor ligand superfamily member Osteoprotegerin ligand CD254 antigen 151190002H23Rik regulator of cell cycle regulator of cell cycle bA157L14.2RGC32 Response gene to complement 32 protein C13orf15 chromosome 13 openreading frame 15 RGC-32 regulator of cell cycle RGCC 16 Sema7asemaphorin 7A, GPI membrane anchor (John Milton Hagen blood group) 17Nrgn neurogranin (protein kinase C substrate, RC3) 18 Gucy1a3 guanylatecyclase 1, soluble, alpha 3 19 Crabp2 cellular retinoic acid bindingprotein 2 20 Bach2 BTB and CNC homology 1, basic leucine zippertranscription factor 2 21 Nm1 N-myc (and STAT) interactor N-myc (andSTAT) interactor N-myc-interactor N-myc interactor Nmi N-myc and STATinteractor 22 Pdk2 pyruvate dehydrogenase kinase, isozyme 2 23 Dgkzdiacylglycerol kinase, zeta 24 Mtss1 metastasis suppressor 1 25 Cd74CD74 molecule, major CD 74 molecule, major histocompatibility complex,histocompatibility complex, class II invariant chain class II invariantchain CD 74 antigen (invariant polypeptide of major histocompatibilitycomplex, class II 26 Ptgfm 27 Klf9 kinesin family member 9 28 Rai14retinoic acid induced 14 29 Lag3 lymphocyte-activation gene 3 30 Slc17a6solute carrier family 17 (sodium-dependent inorganic phosphatecotransporter), member 6 31 Trpm4 transient receptor potential cationchannel, subfamily M, member 4 32 E130308A19Rik uncharacterized proteinKIAA1958 KIAA1958 33 Ryr1 ryanodine receptor 1 (skeletal) 34 A2bp1 RNAbinding protein, fox-1 homolog (C. elegans) 1 35 Pros1 protein S (alpha)protein S (alpha) 36 Ece1 endothelin converting enzyme 1 37 Olr1oxidized low density lipoprotein (lectin-like) receptor 1 38 Stk32cserine/threonine kinase 32C 39 Exph5 exophilin 5 exophilin 5 KIAA0624SLAC2B exophilin-5 SLAC2-B synaptotagmin-like homologue lacking C2domains b Slp homolog lacking C2 domains b SlaC2-b Synaptotagmin-likeprotein homolog lacking C2 domains b 40 Jazf1 JAZF zinc finger 1 41 Nelfnasal embryonic LHRH factor 42 Ddx11 DEAD/H (Asp-Glu-Ala- probableATP-dependent Asp/His) box helicase 11 RNA helicase DDX11 43 Repin1replication initiator 1 44 Cib2 calcium and integrin binding familymember 2 45 Tspan5 tetraspanin 5 46 Cish cytokine inducible SH2-containing protein 47 Ttc3 tetratricopeptide repeat DCRR1 domain 3RNF105 TPRD TPRDIII Tetratricopeptide repeat protein 3 RING fingerprotein 1052 TPR repeat protein D TPRDI TPRDII tetratricopeptide repeatprotein 3 (TPR repeat protein D)11 E3 ubiquitin-protein ligase TTC3 EC6.3.2.— Protein DCRR1 48 Arl3 ADP-ribosylation factor-like 3 49 Fam195afamily with sequence similarity 195, member A 50 Slc13a3 solute carrierfamily 13 (sodium-dependent dicarboxylate transporter), member 3

All of the methods disclosed and claimed herein can be made and executedwithout undue experimentation in light of the present disclosure. Whilethe compositions and methods of this invention have been described interms of preferred embodiments, it will be apparent to those of skill inthe art that variations may be applied to the methods and in the stepsor in the sequence of steps of the method described herein withoutdeparting from the concept, spirit and scope of the invention. Morespecifically, it will be apparent that certain agents which are bothchemically and physiologically related may be substituted for the agentsdescribed herein while the same or similar results would be achieved.All such similar substitutes and modifications apparent to those skilledin the art are deemed to be within the spirit, scope and concept of theinvention as defined by the appended claims.

REFERENCES

The following references, to the extent that they provide exemplaryprocedural or other details supplementary to those set forth herein, arespecifically incorporated herein by reference.

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The invention claimed is:
 1. A method for treating a patient withimmunotherapy comprising administering immunotherapy to the patientafter the patient is identified as having non-anergic T-cells aftermeasuring the level of expression of at least Nrn1, CRTAM, and BACH2 inT-cells from the patient and identifying expression differences of atleast Nrn1, CRTAM, and BACH2 compared to the levels of expression inanergic-T cells.
 2. The method of claim 1, wherein the patient is acancer patient.
 3. The method of claim 1, wherein the immunotherapycomprises a cell-based immunotherapy.
 4. The method of claim 1, whereinthe immunotherapy comprises antibody therapy.
 5. The method of claim 1,wherein the immunotherapy comprises a cancer vaccine.
 6. The method ofclaim 1, wherein identifying a difference in levels of expression of atleast Nrn1, CRTAM, and BACH2 compared to anergic-T cells comprisescomparing the levels of expression to the levels of expression in acontrol or reference of non-anergic T cells.
 7. The method of claim 1,wherein identifying a difference in levels of expression of at leastNrn1, CRTAM, and BACH2 compared to anergic-T cells comprises comparingthe levels of expression to the levels of expression in a control orreference of anergic T cells.
 8. The method of claim 1, wherein thelevels of expression are measured using probes or primers specific forNrn1, CRTAM, and BACH2.
 9. The method of claim 1, further comprisesmeasuring the expression levels of at least 2 more genes from Table 2.10. A method for treating a cancer patient comprising administeringantibody therapy to the patient after the patient is identified ashaving non-anergic T-cells based on measuring the level of expression ofat least Nrn1, CRTAM, and BACH2 in T-cells from the patient andidentifying increased levels of expression of at least Nrn1, CRTAM, andBACH2 compared to the levels of expression in anergic-T cells.
 11. Themethod of claim 10, wherein the patient is identified as havingnon-anergic T-cells based on measuring a level of expression of at leastadditional two more genes from Table
 2. 12. The method of claim 1,wherein the levels of expression are measured using probes or primersspecific for Nrn1, CRTAM, and BACH2.